; Cyclic GMP and guanylate cyclase mediate lipopolysaccharide
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Cyclic GMP and guanylate cyclase mediate lipopolysaccharide


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									Cyclic GMP and guanylate                                                                                                                  cyclase                                   mediate
lipopolysaccharide-induced                                                                                                                Kupffer                                   cell tumor                                                           necrosis
factor-a synthesis
                            Brian            G.         Harbrecht,                            Stewart                C. Wang,                       Richard                L. Simmons,                                             and             Timothy                          R. Billiar
                            Department              of      Surgery,                  University             of     Pittsburgh,                 Pittsburgh,             Pennsylvania

Abstract:         Tumor                       necrosis                   factor-a               (TNF-a)                is an           impor-                  release                has            been            conducted                        in        the         hope         of     modifying                         TNF-a
tant     mediator      in                    sepsis                and        septic              shock.              Kupffer             cells                synthesis                      in      a therapeutic                            fashion.                     TNF-a              production                         is regu-
( KCs)       are the resident           macrophages            of the liver        and are                                                                     lated   at both   the transcriptional         and                                                                 posttranscniptional                                  level
potent        producers        of TNF-a         in response          to inflammatory                                                                           [1, 2]. It is also regulated          by a number                                                                    ofcytokines                             and      other
stimuli        such     as bacterial      endotoxin         or lipopolysaccharide                                                                              bioactive                  compounds                           that                 are     produced      during        the stress     or
(LPS).        Although         the effects       of exogenous          cytokines        such                                                                   septic                 response,                        including                       intenferon--y        [7],     a-adrenengic
as interferon-fly            on TNF-a           production           by macrophages                                                                            agonists                      [8],           prostaglandins                                [9],   and   glucocorticoids            [10].
have       been     fairly    well   studied,       the intracellular            pathways                                                                      Thus,                 the            synthesis                      and            release       of TNF-a         appear       to be a
regulating          KC TNF-a          synthesis        are   largely     unknown.         We                                                                   tightly               controlled                        segment                      of     the          host          immune                       response                and
investigated            the role     of guanylate          cyclase      and      cGMP       in                                                                 are    subject      to regulation         by a number           of mediators.
LPS-induced         KC TNF-a       synthesis.      Exogenous         8-BrcGMP                                                                                      Although         a number        of regulating        factors      governing         TNF-
and dbcGMP           increased    LPS-stimulated           TNF-a        synthesis                                                                              a production             have   been     identified,     the intracellular             signals
but had     no effect       on KC TNF-a          in the absence            of LPS.                                                                             and     second        messenger        systems       mediating          LPS-stimulated
Sodium      nitroprusside        (SNP),        a nitric      oxide-releasing                                                                                   TNF-a          synthesis        are    incompletely            understood.           Protein
substance          that    stimulates             guanylate            cyclase,          increased                                                             kinase        C activity      has been        shown                                                            to regulate                          TNF-a         gene
TNF-a        synthesis       in response             to LPS,         whereas          methylene                                                                transcription           in munine      penitoncal                                                             macnophages                            [ii]. Also        in
blue      and      LY83583,            guanylate            cyclase          inhibitors,            de-                                                        munine         peritoneal        macrophages,                                                                 the   inhibitory                            effects      of
creased        KC      TNF-a         synthesis.           The      inhibitory              effect      of                                                      cyclic           adenosine                           monophosphate                                      (cAMP),                      either            directly              ap-
methylene            blue      could         be     overcome              with        exogenous                                                                plied            or        as a result                              of stimulation                               with            PGE2,                  on TNF-a
dbcGMP          or SNP.       Our results            demonstrate               that guanylate                                                                  mRNA                     accumulation                                 and protein                            secretion             have                been estab-
cyclase      and cGMP            mediate         LPS-induced               KC TNF-a               syn-                                                         lished     [12,  13].  Little      is known,     however,     about    other    second
thesis     and    suggest       that     agents        that    alter      cyclic      nucleotide                                                               messenger        pathways         governing      Kupffen     cell (KC)      or macro-
metabolism           in KCs        may       affect      the response             of these        cells                                                        phage      TNF-a      production.           KCs  are the largest     population          of
to inflammation               and       inflammatory                stimuli.         J. Lcukoc.                                                                fixed   tissue    macrophages             in the body     [14] and     possess     func-
Biol.     57: 297-302;            1995.                                                                                                                        tional               characteristics                                 that           distinguish                         them             from              penitoneal
                                                                                                                                                               macrophages           [14, 15]. They      line the hepatic       sinusoid,                                                                                        are cx-
Key           Words:             cyclic       nucleotides                   .     sepsis             cytokines
                                                                                                #{149}                    .   second             mes-          posed     to portal-derived          antigens      and     endotoxin,                                                                                           and     arc
sengers                                                                                                                                                        potent      producers       of TNF-a         [16]. KCs     have     been                                                                                       hypothe-
                                                                                                                                                               sized            to           play             a     pivotal                 role           in         the        response                     of      the         host            to
                                                                                                                                                               invasive                      infection                      [16]            and            measurements                                 of          hepatic               vein
INTRODUCTION                                                                                                                                                   TNF-a        levels     have     prompted         the suggestion          that  liver    and
                                                                                                                                                               gut TNF-a           production        accounts        for at least one-fourth         of the
Tumor       necrosis                  factor-a      (TNF-a)      has been        hypothesized                                                        to        total    body       TNF-a       production         following      endotoxemia         [17].
be one        of the                  central      mediators       in the     pathophysiologic                                                                      The    second        messenger        cyclic       guanosine      monophosphate
events    following                    endotoxemia           and sepsis   in both       expenimen-                                                             (cGMP)                        is produced       by the enzyme                                                     guanylate         cyclase     and is
tal animals        and                humans        [1]. It is a multifunctional             cytokinc                                                          responsible                       for a variety    ofccllulan                                                 effects     including         the phos-
produced           by activated       macrophages             that    is secreted        as a                                                                  phorylation                               of          cellular                     proteins                     through                    the           actions                   of
 17-kDa       protein   in response        to bacteria       or bacterial      endotoxin                                                                       specific                protein                    kinases                 [18].          Some               ofthe            effects               ofcGMP                   are
(lipopolysacchanide,            LPS).      Exposure        of macrophages           to LPS
results       in a prompt         increase        in TNF-a           messenger         RNA
(mRNA)            and TNF-a       synthesis,        followed       by a rapid       decline
in      measurable                TNF-a                 activity                [1,     2].     In       addition             to      TNF-a,                         Abbreviations:                         TN        F-a        , tumor           necrosis            factor         a ; LPS,           lipopolysaccharide;
                                                                                                                                                               N0,                  nitric           oxide;            cAMP,                adenosine                  3:5-cyclic                   monophosphate;                          KC,
activated        macrophages            produce         a number         of other        inflam-
                                                                                                                                                               Kupffer              cell;          cGMP,             guanosine                3 :5-cyclic                   monophosphate;                          MB,        methylene
matony        mediators           in response            to LPS,         including          inter-                                                             blue;       LY83583,                      6-anilino-5,8-quinolinedione;                                                SNP,          sodium            nitroprusside;
leukin-1,       intenleukin-6,          prostaglandin            E2 (PGE2),          and     nitric                                                            LDH,    lactate     dehydrogenase;                                             L-NAME,                   N-nitro-1.-arginine                            methyl    ester;
oxid       (N0)            [3, 4]. These           mediators       together         produce           a                                                        dbcAMP,      N6,2’-O-dibutyryladenosine                                                          3:5-cyclic        monophosphate;                            dbcGMP,
complex         integrated        response         to an infectious           stimulus.                                                                        N2,2’-O-dibutyrylguanosine                                                  3’ :5-cyclic                 mnonophosphate;                             8-BrcGMP,                     8-
                                                                                                                                                               bromogctanosine                              3’ :5-cyclic                 monophosphate;                             iNOS,            inducible              nitric        oxide
     Because        ofTNF-a’s         central       role in the host response                to an
                                                                                                                                                               synthase;                    NMA,                  N’-monomethyl-t.-arginine;                                            PGE2,                prostaglandin                    E2;
inflammatory             stimulus      and      septic    shock,     therapies        based        on
                                                                                                                                                               PBS,        phosphate-buffered                                     saline.
the        inhibition           ofTNF-a                   secretion                   or neutralization                            of TNF-a                          Reprint                 requests:               Brian           G.       Harbrecht,                     Department                      of     Surgery,             AlOlO
activity             have      been          proposed                     [5,         6].     Considerable                         investiga-                  Presbyterian                        University                Hospital,               DeSoto             at O’Hara,                   Pittsburgh,               PA        15213.
tion,         therefore,              into        the       regulation                        of TNF-a                secretion                  and                 Received                 September                     7,     1994;          accepted              October               24,       1994.

                                                                                                                                                          Journal              of      Leukocyte                         Biology                         Volume                 57,      February                    1995                   297
                                                                                                                                                                               .             .     .    .                           (control                 -      experimental)
directly              opposite            to      those           of cAMP,               which,             as      noted             above,
                                                                                                                                                                       %       inhibition                             =                                                                                       x 100
is      a   potent               down-regulator                          of     TNF-a                 synthesis                [12,         13].
However,         the function       of cGMP           in KC cytokine             synthesis      is
largely     unknown.          In this study,           we investigated            the role of                                                               The        ability                   ofcultune                      supernatants                            to induce                 L929            cytotoxic-
cGMP          in     KC      TNF-a          synthesis         utilizing         membrane-                                                                   ity       could              be            completely                           neutralized                       with           polyclonal                     rabbit
permeable          analogues       of cGMP           directly      applied       to cultured                                                                anti-mouse                          anti-TNF-a                                  serum                (Genzyme),                        demonstrating
KCs.      We also studied            the role of this second                 messenger         in                                                           that     this                    bioassay      was                              specific               for TNF-a                         and     was                    not
TNF-a        synthesis        by culturing            KCs     with       methylene         blue                                                             affected                by            the presence                                 of     other cytokines                               in the               culture
(MB)      and LY83583,           inhibitors        of guanylate          cyclase      [18, 19],                                                             supernatant.                            L929   viability                                 was not affected                              by the               concen-
and         with          the       N0-neleasing                              agent            sodium               nitnopnusside                           trations                 of            MB,     nitnoprusside,                                           LY83583,        LPS,                           or        cyclic
(SNP),                a   stimulator                  of     guanylate                cyclase            [20].          The           results               nucleotide                           analogues       used     in                              these        experiments.
demonstrate                       that          guanylate               cyclase                 and        cGMP                 regulate
LPS-induced                       KC           TNF-a              synthesis.                                                                                KC viability
                                                                                                                                                            KC viability          after    culture     was determined                                                                         by the release            of
                                                                                                                                                            lactate      dehydrogenase           (LDH)     into culture                                                                      supernatants            [4].
MATERIALS                         AND            METHODS
                                                                                                                                                            LDH        levels    were    determined       utilizing        an                                                               automated           proce-
                                                                                                                                                            dune      on      a Technitron          RA500        autoanalyzer                                                                        (Technitron,
KC isolation
                                                                                                                                                            Tarrytown,          NY).
KCs     were     isolated      from     male  Sprague         Dawley      rats     (250-
300 g) utilizing          an in situ liven perfusion          with 0.2%        pronase                                                                      Materials
E [4]. After       perfusion,       the liven was excised,        minced,       and in-
                                                                                                                                                            LY83583             was purchased               from       Calbiochem           (La Jolla,        CA).
cubated      for 40 mm in a continuously              stirred     bath    containing
                                                                                                                                                            LPS        (from       E. coli Oiil:B4),              MB,        SNP,      N-nitro-L-arginine
0.2%     pronase         E with DNAse              added       at 20-mm          intervals.         The
                                                                                                                                                            methyl         ester    (L-NAME),               actinomycin           D, N6,2’-O-dibutyryl-
solution       was filtered           through        mesh       gauze       and     centrifuged,
                                                                                                                                                            adenosine            3’ :5-cyclic         monophosphate                (dbcAMP),            N2,2’-O-
and then        the KCs were              isolated      from      the resuspended                pellet
                                                                                                                                                            dibutyrylguanosine                 3’ :5-cyclic       monophosphate              (dbcGMP),          and
by centrifugal           elutniation.         The    resulting        cell suspension              con-
                                                                                                                                                            8-bromoguanosine                    3’ :5-cyclic        monophosphate                (8-BrcGMP)
sisted   of85-95%              KCs,      as demonstrated              by penoxidase              stain-
                                                                                                                                                            were       purchased           from      Sigma        (St.     Louis,     MO).
ing. Viability          exceeded          96%      by trypan         blue      exclusion.          KCs
were     resuspended               in Williams           medium           E (Gibco,           Grand
Island,     NY) containing                 15 mM       HEPES,           10 U/L          penicillin,
 100 mg/L        streptomycin,             2 mM       L-glutamine,             10 mM        insulin,
and 5% calfserum                  at a concentration             of5      x 10 KC/ml,                and
were cultured           in 16-mm           wells at 1 ml/well.             After     4 h, nonad-
henent     cells were         removed         by washing          with warm           phosphate-
buffered           saline       (PBS)        and       the medium              was replaced.              After
overnight            incubation,            the KCs were again                    washed        with warm
PBS        and      then      the experimental                  conditions            were      established
in medium                 containing            2.5%        calf serum.              At the indicated
time        points,        culture         supennatants               were       collected         and      cen-
tnifuged         and the cell-free                 supennatant             was frozen            at - 70#{176}C
prior        to assay.          All culture             conditions           were        represented              in                                            C
triplicate.           Data       represent          the mean             ± SEM.          Student’s         t-test                                               0             40
was used             to determine              statistical         significance;            P < .05 was
considered                  significant.
Measurement                            of TNF-a                                                                                                             co>
                                                                                                                                                            -         3JJ
TNF-a                 activity            in      culture                supernatants                     was           determined                              CLl
using        the L929                  fibroblast  assay                      [4].     Briefly, L929                      fibnoblasts
were         cultured                  at a concentration                               of S x 10                        cells/mI               in
96-well       culture      trays      at 0.1 ml/well           in the presence            of serial                                                         3.
dilutions         of KC culture           supernatant            and     1 tg/ml      actinomy-
cm D. After           20 h, the degree             of cell lysis was determined                      by
staining        the cells with 0.5%             crystal      violet     and the absorbance
measured           at 550 nm using             an automated              microplate          reader.
The       quantity       of TNF-a            was      determined           by extrapolation
from       a standard            curve      constructed             for each        experiment
utilizing         known       quantities         of recombinant               munine        TNF-a                                                                                        0                       i-                         10.8                                      i0                    i-
(rmTNF-a;             Genzyme,           Boston,        MA).        The    percent        stimula-
tion produced            by the experimental                 conditions        was calculated
                                                                                                                                                            Fig.       1. Effect                 of8-BrcGMP                         and dibutyryl-cyclic                         GMP                   LPS-induced                   KC
using           the       following              formula:
                                                                                                                                                            TNF-cr          synthesis.                  Kupffer                cells        were        incubated             with     I jsg/ml            lipopolvsaccha-
                                                                                                                                                            ride      and     the         indicated                   concentrations                      of8-BrcGMP                      (E)      and      dbcGMP                  (s).
                                                           ( experimental                  -      control)                                                  Culture  supernatants                                  were           collected              3 h following                the       addition           of LPS            and
            %         stimulation                 =                                                                       x 100
                                                                                                                                                            analyzed   fr TNF-a                                  activity              by    the     L929        fibroblast           bioassay.            Data         represent
                                                                               control                                                                      the mean     ± SE,1                             of    triplicate                 cultures            from         three         separate             experiments.
                                                                                                                                                            LPS-stimulated                             KCs        (control)                 produced             220      ± 31        U      TNF-a/ml.                  P       <     .05
The         percent              inhibition                 was      calculated                  using            the     formula:                          compared                to       untreated                    control             KCs.

 298                  Journal            of Leukocyte                    Biology                Volume             57,        February               1995
RESULTS                                                                                                                                                                                                                                                                                                                                                       **

To test                 the               hypothesis                         that         cGMP                   regulates                    KC               TNF-a                 syn-
thesis,                 KCs                     were               cultured                   with              the             membrane-permeable
cGMP                     analogues                               dbcGMP                       and             8-BrcGMP         and stimulated
with            LPS.          Culture                              supennatants                               were   collected    3 h after   the
addition                           of         LPS            and            analyzed                     for       TNF-a                     activity                   (Fig.              1).
Both               cGMP                         analogues                          increased                    KC              TNF-a                   in response                         to
LPS             with                    maximal                      effect             produced                      by         10          M          dbcGMP                        and
i08             M          8-BrcGMP.                                   Unstimulated                                   (non-LPS-exposed)                                              KCs
produced                            less             than            1 U            TNF-a/ml,                         whereas                      LPS-stimulated
(control)                   KCs produced       220     ± 31 U TNF-a/ml            after  3 h of                                                                                                      -a
exposure                    to LPS.  When      supernatants        were collected       6 h fol-                                                                                                     -C
lowing                    LPS,   the  increase      in LPS-stimulated           TNF-a      pro-
duced                 by            8-BrcGMP                                 persisted                   (10               M,         54       ±          20%            stimula-                         C
tion;      i0                            M,  102 ±                          15%            stimulation;                               10            M,            64         ±      23%
stimulation;                             cGMP
                                         all P <   analogues        did not lead
                                                                            .05).    to
detectable                      in TNF-a
                                      increases in KCs       not exposed      to LPS
(medium                             alone:
                                 ± 0.3    U TNF-a/ml;
                                                0.9             dbcGMP,       10   M:
0.8    ± 0.3     U TNF-a/ml;             n = 6,    P = NS).         When      10    M
8-BrcGMP           was    cultured         with   LPS-stimulated           KCs,    in-                                                                                                                                      0
creased      TNF-a     synthesis       compared      with     that of control    KCs
was           detectable                              out            to 24 h (data                         not          shown).                    When                  cultured
with               10                   M        dbcAMP,                            LPS-stimulated                                     KCs                produced                     less
than            50%                       of the TNF-a                                 of non-cAMP-exposed                                                       KCs        (data                                      -20
not           shown),                        consistent                             with   findings     of other                                              investigators                                                     I 0.8                              io7                             10.6                                                           1
[12,           13].
    The       increase        in                                        LPS-stimulated               KC      TNF-a      synthesis                                                                                                                                                                         M
could      also be produced                                                  by culturing           KCs     in the presence         of                                                                Fig.      2. Methylene                      blue      and        LY83583               inhibit        Kupffer             cell tumor             necrosis           fac-
increasing         concentrations                                               of SNP,         an NO-nelcasing               agent                                                                   tor      synthesis.            Kupffer               cells      were        incubated                with      I sg/ml               LPS       and      the       mdi-
that           stimulates                                 guanylate               cyclase    [20]    (Table     1). SNP      alone,                                                                   cated        concentrations                     of methylene                    blue         (U)     and     LY83583                 (ES).     Supernatants

in      the         absence                     ofLPS,                        did not result      in increased       KC TNF-a                                                                         were       collected            after        3 h and             analyzed              for     TNF-a          activity               by the      L929           bioas-
                                                                                                                                                                                                      say.      Data         represent              the       mean            ± SEM           of    one       of   three         representative                     experi-
(medium                                  alone:       0.8                     ± 0.3       U TNF-a/ml;           SNP,      1 mM:
                                                                                                                                                                                                      ments.           P        <     .05;         **J.5      <       .01 compared                       to untreated                 control           KCs.           LPS-
1.6        ±        0.6                 U       TNF-a/ml;                        n = 6, P = NS).                                                                                                      stimulated                (control)             KCs           produced             189         ±     14 U      TNF-a/ml.
    To further                                   evaluate                    the        effect           of      endogenous                                  guanylate                     cy-
clase  activity                                  on          KC             TNF-a                synthesis,                       KCs              were              incubated
with               increasing                               concentrations                                of      the            guanylate                        cyclase                  in-
                                                                                                                                                                                                          To           further                 document                      that                  the   effect                 of         MB           on LPS-
hibitons    MB     [18] and     LY83583          [19] (Fig.     2). Both     MB and
                                                                                                                                                                                                      induced               KC                TNF-a                    synthesis                     was    due                 to         the        actions   of
LY83583       produced       concentration-dependent                  decreases         in
                                                                                                                                                                                                      cGMP,                  KCs              were                 cultured                  with           MB             in        the           presence                    of
supernatant       TNF-a     levels      when        added    with   LPS.     MB     and
                                                                                                                                                                                                      dbcGMP                   and             SNP                   and      stimulated                           with       LPS  (Table         3).
LY83583      had     no measurable           effect      on KC TNF-a         levels     in
                                                                                                                                                                                                      When              cultured               with                  KCs     individually,                           MB decreased           while
the absence       of LPS     stimulation           (data    not shown).       The     in-
                                                                                                                                                                                                      dbcGMP                  and             SNP                  increased        TNF-a                          synthesis.     The     inhibi-
hibitony      effect   of MB on KC TNF-a            was evident     at LPS con-
                                                                                                                                                                                                      tory         effect           of       MB             on        KC         TNF-a                    could            be     overcome                     by        the
centrations          as low    as 10 ng/ml      and    as high     as 10 jg/ml
                                                                                                                                                                                                      addition                  of either                   dbcGMP                      on SNP.
(Fig.     3). Likewise,      the inhibitory    effect   of LY83583      persisted
                                                                                                                                                                                                          The           production                          of N                 0 is associated                              with activation                            of
through       the same      LPS concentrations        (data   not shown).        The
                                                                                                                                                                                                      soluble            guanylate                         cyclase               and increases                             in cGMP      [21].                         KCs
decrease      in TNF-a                                             produced        by MB and LY83583          could   not
                                                                                                                                                                                                      possess               the          inducible                     N0                    synthase               (iNOS)                    enzyme                   [22]
be attributed        to                                          changes         in KC    viability,  because       LDH
                                                                                                                                                                                                      and    produce   significant        amounts      of N      0 in response             to
release    into culture                                             supennatants       was not increased        by these
                                                                                                                                                                                                      inflammatory     stimuli       [4]. To determine       whether      endogenous
agents    (Table     2).
                                                                                                                                                                                                      KC N = 0 production               was involved      in these    findings,         KCs
                                                                                                                                                                                                      were    cultured  with       LPS    and    L-NAME,       a specific       inhibitor
 TABLE                     I   .            Effect          of     Sodium               Nitroprusside                      on         LPS-Stimulated                             Kupffer              of NOS,          and supernatants                                            were      collected     3 h following         the
                                                                        Cell          TNF-a              Synthesis                                                                                    addition          of LPS.    KCs                                          exposed         to LPS      alone     produced
                                                                                                                                                                                                      137         ± 12 U TNF-a/ml,                                             and     those      cultured    with    LPS      plus
          toNi          SNP’                                                                                                                 Percent             stititulation                        2 mM              L-NAME                            produced                  131 ± 8 U TNF-a/ml              (n = 2 ex-
                                                                                                                                                                                                      peniments,         P                    =      NS).                Similarly,                      when          KCs                 were            cultured
          0.0                                                                                                                                          0.0       ±     3.72
                                                                                                                                                                                                      with    LPS     and                         NGmonomethylLarginine                                                       (NMA,                       2 mM),
          0.01                                                                                                                                     21.5          ±     8.2k
          0.1                                                                                                                                      26.8          ±
                                                                                                                                                                                                      another     inhibitor                         of NOS,       no differences                                           in TNF-a                     synthesis
          1.0                                                                                                                                      52.1          ±      9.2’                          could          be detected                        compared                             to      control     KCs   cultured                                       with
                                                                                                                                                                                                      LPS          alone   after                      3 h of culture                               (data     not shown).
       “After           overnight                         culture,            Kupifer            cells         were         washed             and            stimulated               with
LPS           (Isg/rnl)                       in the presence                        of the indicated                       concentrations                       of SNP.      Cul-
ture       supernatants                           were   collected                      after  3 h and                      analyzed                for       TNF-a      content
I))     the         L929                 assay        .     Data        represent                the      mean              ± se              of       three         separate              cx-
periments.                         LPS-stimulated                           (control)            KCs           produced                256         ±         27 U TNF-a/ml.
       “P <             .05             vs.      0 mM                SNI.                                                                                                                             TNF-a       has been                            hypothesized                         to be an                  essential                     mediator                   in
       ‘P <             .001              vs.     0 taM               SNP.                                                                                                                            the   deleterious                              effects      of                  endotoxemia                        and                 in      endotoxin

                                                                                                                                                                         Harbrecht               et al.        Cyclic               GMP             reduces                   Kupffer               cell        TNF-cs               synthesis                           299
                                                                                                                                                                                                                              TABLE                    3.        cGMP              and       Sodium              Nitroprusside                       Overcome                     the         Inhibition
                                                                                                                                                                                                                                                             of TNF-a                 Synthesis               Produced                by     Methylene                       Blue

                                                                                                                                                                                                                                  Condition”                                                                                               Percent          of    contr(       )l    I’NF-a               release

                                                                                                                                                                                                                                  Medium                                                                                                                          100         ±      2
                                                                                                                                                                                                                                  MB, 0.1                    mM                                                                                                    47         ±      35
                                                                                                                                                                                                                                  dbcGMP,                     1 gM                                                                                                115         ±      75
                                                                                                                                                                                                                                  SNP,                I mM                                                                                                        134         ±
                                                                                                                                                                                                                                  MB             +      dbcGMP                                                                                                      69        ±
                                                                                                                                                                                                                                  MB+SNP                                                                                                                            88±                 12’

                                                                                                                                                                                                                                  “After               overnight             culture,               Kupffer             cells      were         washed                 and          cultured                 with
                                                                                                                                                                                                                             either         methylene                   blue        (MB),            dibutyryl               cyclic         GMP          (dbcGMP),                            or     sodium
                 300                                                                                                                                                                                                         nitroprusside                      (SNP)          at the        indicated              concentrations.                    LPS         (I jig/mI)                 was         added
                                                                                                                                                                                                                             to induce                  TNF-o            synthesis.                After       3 h,        supernatants                 were            collected                  and       ana-
U.                                                                                                                                                                                                                           lyzed         for         TNF-cr           content.             Data          represent            the    mean             ±        sEu         of three               separate
z                                                                                                                                                                                                                            exrriments.                      LPS-stimulated                       KCs        (control)           produced             223          ±        34 U TNF-a/ml.
                                                                                                                                                                                                                                   ‘P <               .05    vs.      medium.
                 200                                                                                                                                                                                                              ‘P <                .001      vs.     MB          alone.

                 100                                                                                                                                                                                                              In this report,       we demonstrate              that    cGMP    and                                                                                        endoge-
                                                                                                                                                                                                                             nous    guanylate       cyclase    activity      regulate        KC TNF-a                                                                                          synthe-
                                                                                                                                                                                                                             sis.   Membrane-permeable                   cGMP         analogues    were                                                                                         able    to
                                                                                                                                                                                                                             augment       KC TNF-a          production,         in contrast     to the                                                                                       suppres-
                                                                                                                                                                                                                             sion      ofTNF-a          produced           by cAMP        analogues          previously
                                                                                                                                                                                                                             described      by other        investigators         [12, 13]. The        augmentation
                                                                                                                                                                                                                             ofTNF-a        synthesis        was greaten         for 8-BncGMP            compared          to
                                                                                                        LPS,                   ug/mi
                                                                                                                                                                                                                             dbcGMP         and     was present             at relatively      low concentrations
Fig.         3. Methylene                                blue          inhibits                    Kupifer              cell      tumor              necrosis                 factor            synthesis                    (105_108       M). This         effect     was also present          at both     early     and
at       diflerent                     concentrations                                of            lipopolysaccharidc.                                 Kupffer                  cells           were            in-
                                                                                                                                                                                                                             late time    points      in culture.         The addition       ofexogenous           cGMP
cubated                    with            LPS            alone             (LI)          or        with          0.1       mM               methylene                 blue              (U)      and          the
                                                                                                                                                                                                                             is not sufficient        to initiate        KC TNF-a         synthesis,       however,        as
indicate(l                      concentrations                              of LPS.                     Supernatants                         were        collected                  after         3 h and
analyzed                        for        TNF-a                  activity                     by         the       L929               bioassay.               Data                represent                   the           cGMP      analogues          were      unable     to result    in measurable            TNF-
mean               ± SENt                  from           one          of       three               separate              experiments.                         *,D        <        .01         compared                      a synthesis      in the absence                                                  of LPS.     This     finding       is in contrast
tO      LPS            alone.                                                                                                                                                                                                to the activity     in penitoneal                                                  macnophages,            where      cGMP        ana-
                                                                                                                                                                                                                             logues  were able to stimulate                                                       TNF-a       synthesis       in the absence
                                                                                                                                                                                                                             of LPS     [23].    The    reason                                                 for this differential            sensitivity         to
                                                                                                                                                                                                                             cGMP     analogues       in these                                                separate       macrophage            populations
shock       [1, 2]. Neutralization             ofTNF-a            with anti-TNF-a                                                                                                               anti-
                                                                                                                                                                                                                             is       unclear.
bodies        ameliorates         many       of the pathophysiologic                                                                                                                   effects        of
                                                                                                                                                                                                                                  The       activity         of guanylate            cyclase      mediated           the    LPS-
LPS      and improves           survival       in experimental               endotoxin                                                                                                        shock
                                                                                                                                                                                                                             induced         KC TNF-a               synthesis     in these      experiments.            MB and
models         [5, 6]. This      finding      has prompted              efforts       to                                                                                              inhibit        or
                                                                                                                                                                                                                             LY83583,             inhibitors           of guanylate          cyclase,        decreased        KC
neutralize          the damaging           effects     of TNF-a            during                                                                                                    endotoxe-
                                                                                                                                                                                                                             TNF-a         synthesis.          This      effect  was not due to altered                  KC via-
mia or septic             shock    and has resulted              in efforts         to                                                                                               better       un-
                                                                                                                                                                                                                             bility     because           LDH         release    in culture         medium,          a method
derstand          the cellular        mechanisms             involved         in the                                                                                                   synthesis
                                                                                                                                                                                                                             that     correlates          well with          the ability     of these       cells to exclude
and release           ofTNF-a         [6]. Both      transcriptional               and                                                                                                 posttnan-
                                                                                                                                                                                                                             trypan       blue       [4], was unchanged                by these     conditions.          We can-
scniptional          control    of TNF-a           synthesis         have       been                                                                                                  described
                                                                                                                                                                                                                             not         exclude                      the          possibility,                      however,                  that              MB            or         LY83583
I 1, 2, 6]. However,                                           the                 precise                      cellular  mechanisms                                                 that         occur
                                                                                                                                                                                                                             may           have               injured                 the          KCs          in       a manner                     that             does              not             result
following                             LPS                binding                     to cell                    membranes        and                                  result                   in rapid
                                                                                                                                                                                                                             in        LDH                   release.                 It      is      also             possible                that              significant                             LDH
TNF-a                           synthesis                         and              release                       are           incompletely                                understood.
                                                                                                                                                                                                                             release     may       not occur     at the early       time   points     studied        in
                                                                                                                                                                                                                             these    experiments.         However,       the finding    that     the inhibitory
                                                                                                                                                                                                                             effect    of these       compounds        could    be overcome          with     either
       ‘FABLE                     2.             Eflect           of        Methylene                           Blue        and              LY83583                 on        Kupffer                 Cell                  SNP     or exogenous         cGMP      suggests    that the decreased          TNF-a
                                                                                           LDH                  Release”                                                                                                     synthesis                       produced                        by       MB             and          LY83583                        was          due              to          their
                                                                                                                                                                                                                             effect     on guanylate           cyclase        and not due to permanent                    or ir-
       (:ttdit              .                                                                                                                                                      L_DH          ( I U/I.)                   reversible         changes       in KC viability.
                                                                                                                                                                                                                                  Exogenous            cGMP         and      SNP      were      unable       to completely
       Medium,                         n        =       15                                                                                                                         12.7          ±      1.7
                                                                                                                                                                                                                             overcome          the inhibitory           effect   of MB on LPS-induced                     TNF-
       LPS,            n         =         17                                                                                                                                      15.7          ±      1.4
       LPS             +         0.01               mM          MB,             mi    =            15                                                                              12.3          ± 2.4
                                                                                                                                                                                                                             a synthesis.           The      reason        for the incomplete                restoration         of
       LIS             +         0.1        mM               MB,            a      = 9                                                                                               6.0         ±      0.5                  TNF-a          synthesis        with       these     agents         is unclean.          MB     and
       LPS             +         0. 1 niM                    LY83583,                          n      = 3                                                                          16.3          ± 0.3                       LY83583          may       have     permanently            altered        the guanylate           cy-
       Freeze-thaw,                                 n    = 9                                                                                                                       93.      1 ±         2.6                  clase     enzyme         on changed          its activity.       It is possible         that  these
                                                                                                                                                                                                                             agents       incompletely            restored       cGMP          levels     on incompletely
        “After              overnight                        culture,                Kupifer                    cells       were              washed            and            incubated                      with           overcame           the inhibitory            affect     on guanylate            cyclase.     Rela-
combinations                               of LPS,                 methylene                            blue       (MB),               and          LY83583                   at     the        indicated
                                                                                                                                                                                                                             tively                  large         concentrations                              of       SNP           and            cGMP                    were              utilized,
concentrations.                                     Supernatants                          were             collected                 after       3 h and              analyzed                   for     LDH
content.                   Separate                      Kupfl#{232}r cells                    were             subjected                to three             rapid           cycles             of freeze-
                                                                                                                                                                                                                             but cGMP                             levels            of KCs                 were            not measured                             in these                        expeni-
thaw             iii       liquid               nitrogen.                   Values                   represent                 the       mean             ±      SEM           of (n)            cultures.                   ments.  It                         is also             possible                that            these  agents                          did not                         provide

 300                            Journal                       of Leukocyte                                      Biology                         Volume                    57,            February                     1995
sufficient            cGMP     at the time     it was required         in the intracellu-                                                                      posed              to MB,                  similar
                                                                                                                                                                                                           a result  to that found      in human                                                                   mono-
lan cascade              of events   following      LPS   stimulation.        The      intra-                                                                  cytes             [36].                     of second
                                                                                                                                                                                                  Activation              messenger       systems                                                                such    as
cellular    events      that occur    following     LPS binding                                                           to the mac-                          cGMP                  may     therefore      account     for the changes           in                                                            cytokine
rophage        membrane         are    incompletely        understood.                                                            TNF-a                        synthesis               produced        by N       0 donors     as documented                                                                    by other
mRNA         accumulation          and secretion     occur      rapidly                                                       following                        investigators                               [36].         Our                findings                suggest                  that        exogenous
stimulation        with     LPS  [1, 2, 10], and the temporal                                                               accumula-                          sources               ofNO                          may          lead         to      increased                KC           TNF-a            synthesis
tion    of cGMP           induced        by these       agents        is unknown.         Addi-                                                                and that    the TNF-a        synthetic       pathway     was not operating        at
tional    studies      will be needed           to correlate         the accumulation           of                                                             maximal       capacity    under        the conditions       studied.     Maximal
cGMP        with     the degree          of TNF-a          synthesis       in this system.                                                                     TNF-a     synthesis    in vivo may therefore             be dependent        on the
     In this study,          we were       able     to decrease          TNF-a       synthesis                                                                 products    of second    messenger         systems,   such as phosphorylated
with    the guanylate           cyclase      inhibitor        MB on LY83583.               How-                                                                proteins,    on the availability         of second    messenger       compounds
even, the use of L-NAME                     and NMA,             agents      that can block                                                                    themselves                         following                    exposure                    to     endotoxin.                        Macrophages
endogenous           N0          synthesis       and result         in decreased        cGMP                                                                   have    been      shown        to have    multiple     steps    of amplification           in
in selected       cell populations            [24],    had no effect.          Our    findings                                                                 TNF-a       synthesis.         LPS leads      to a 100-fold      increase       in TNF-a
suggest             that,   at the                early      time   points    studied                         in these               expeni-                   mRNA         and a 10,000-fold           increase    in protein        production        [1].
ments,             endogenous                      KC       N     0 production                            plays    little             role in                  Our   findings         suggest    that this amplification            process      could   be
TNF-a              synthesis.               This        result      is consistent                          with          the findings                          increased                further      under       the    proper      conditions.       KCs     are
of other             investigators                    who      have    found      no                     effect          with  the in-                         present               in the liver adjacent           to endothelial         cells and hepato-
hibition            of N              0     synthesis      on TNF-a     or interferon                                            produc-                       cytes,             both    cell types     capable       of producing          NO      [22].   Not
tion     by         elicited              munine      penitoneal    macrophages                                              [25].    KCs                      only              is the       liven   an    organ       capable       of producing         large
possess     iNOS      activity     following     exposure        to LPS and the ap-                                                                            amounts            of NO            [22],      but splanchnic                TNF-a          production
pearance       of nitrite      and     nitrate,   the metabolic          end      products                                                                     ( intestine         + liven/spleen)            accounts          for nearly          one-quarter            of
of N       0 metabolism,            takes 4-6 h for minimal              changes        to be                                                                  the TNF-a             produced          by the body             following          intravenous           en-
detectable       with     maximal         NOS   activity      occurring       at 12-24       h                                                                 dotoxin           administration                [17].      Our        experiments               with     the
[4, 26]. In contrast,           the appearance           ofTNF-a        following        LPS                                                                   N        0-releasing           substance           SNP       demonstrate               that      KCs     are
is rapid           under         in       vivo        and         in   vitro         conditions                 [1,     2] with           max-                 able       to increase           TNF-a           synthesis          in the        presence           of cx-
imal     TNF-a         synthesis         occurring         within       2-4 h of LPS        stimu-                                                             ogenous           N0.          This      finding        suggests         that    endothelial           cells
lation     [27].     Thus,        maximal          TNF-a        synthesis        appears   to pre-                                                             on       hepatocytes             may        modulate             KC        TNF-a            production
cede     iNOS       activity        temporally         in rat KCs following              LPS and                                                               through          the stimulation              of KC guanylate                 cyclase         in a para-
would      further        suggest       that iNOS          activity       does not play a role                                                                 cnine       fashion.
in KC TNF-a                 synthesis.                                                                                                                         In summary,            we have demonstrated                    that guanylate          cyclase
     The     presence         of a constitutive,              agonist-stimulated            NO                                                             and     cGMP         regulate         LPS-stimulated              KC TNF-a            synthesis.
synthase         (cNOS)           in KCs         has     not been          demonstrated.         We                                                        Our      results    suggest         that cGMP            is an important           component
cannot       completely             rule out a role for NO                        in KC TNF-a                                                              in the signal         transduction            pathway       that leads      to the synthesis
synthesis,                however.                Guanylate                    cyclase           can       be         stimulated                 by        and release         of TNF-a            following       stimulation        of KCs by LPS.
N=O      at nanomolar                           concentrations                           [18], which     is below                            the           Determining             the mechanism               of action        of cGMP        in produc-
level of detectability                         of the nitrite                        assay     used  to establish                            the           ing increased            KC TNF-a,              whether       at the transcriptional               or
activity   of iNOS                         in KCs      [4, 26].                     Therefore,      we cannot                              con-            posttranscniptional                level,     will require         further    investigation.
elusively            rule       out         the       presence                 of    extremely                  small        quantities
of N       0 that may be produced          despite     the presence      of NOS
inhibitors      and    may   be sufficient         to stimulate       guanylate                                                                            ACKNOWLEDGMENT
cyclase     and result   in TNF-a    synthesis.       However,      our finding
that     two distinct      inhibitors       of NOS       (L-NAME         and      NMA)                                                                     This               work   was                    supported                   by NIH                  grants          GM-37753                        (R.L.S.)
had no effect         on TNF-a        synthesis       makes    this hypothesis       un-                                                                   and               GM-44100                         (T.R.B.).
likely.     Likewise,    in stimulated           munine     macrophages          the ap-
pearance         of iNOS        mRNA          and     iNOS     protein     requires      a
minimum            of4-6        h to develop              [28-30],        correlating         fairly     well                                              REFERENCES
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302                  Journal                       of Leukocyte                              Biology                     Volume                57,       February                      1995

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