Determination of in vivo protein synthesis in human palatine tonsil

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					                                                                               Clinical Science (2005) 108, 179–184 (Printed in Great Britain)   179




                 Determination of in vivo protein synthesis
                                  in human palatine tonsil

Anna JANUSZKIEWICZ∗ , Maria KLAUDE∗ , Karin LORE†, Jan ANDERSSON†,
                                               ´
                               ∗
Olle RINGDEN‡, Olav ROOYACKERS and Jan WERNERMAN∗
          ´
∗
  Department of Anaesthesiology and Intensive Care, Karolinska University Hospital at Huddinge, Karolinska Institutet,
Stockholm 141 86, Sweden, †Department of Medicine, Center for Infectious Medicine, Karolinska University Hospital at
Huddinge, Karolinska Institutet, Stockholm 141 86, Sweden, and ‡Department of Clinical Immunology, Karolinska University
Hospital at Huddinge, Karolinska Institutet, Stockholm S141 86, Sweden


A     B     S     T     R     A      C     T

          The palatine tonsils are constantly exposed to ingested or inhaled antigens which, in turn, lead to
          a permanent activation of tonsillar immune cells, even in a basic physiological state. The aim of
          the present study was to investigate if the immunological activation of the human palatine tonsil
          is reflected by a high metabolic activity, as determined by in vivo measurement of protein synthesis.
          The protein synthesis rate of the tonsil was also compared with that of the circulating T-lympho-
          cytes, the total blood mononuclear cells and the whole population of blood leucocytes. Phenotypic
          characterization of immune-competent cells in tonsil tissue and blood was performed by flow
          cytometry. Pinch tonsil biopsies were taken after induction of anaesthesia in healthy adult patients
          (n = 12) scheduled for ear surgery, uvulopalatopharyngoplasty or nose surgery. Protein synthesis
          was quantitatively determined during a 90-min period by a flooding-dose technique. The in vivo
          protein synthesis rate in the palatine tonsils was 22.8 + 5.7 %/24 h (mean + S.D.), whereas pro-
                                                                   −                   −
          tein synthesis in the circulating T-lymphocytes was 10.7 + 3.4 %/24 h, in mononuclear cells was
                                                                      −
          10.8 + 2.8 %/24 h and in leucocytes was 3.2 + 1.2 %/24 h. CD3+ lymphocytes were the most
               −                                           −
          abundant cell population in the tonsil. The in vivo protein synthesis rate in human tonsils was
          higher compared with the circulating immune cells. This high metabolic rate may reflect the
          permanent immunological activity present in human tonsils, although cell phenotypes and activity
          markers do not explain the differences.




INTRODUCTION                                                       elimination of the invaders. This is reflected by changes in
                                                                   the functional activity of immune-competent cells, such
The immune system is a dynamic network of cells, tissues           as synthesis of cytokines or other regulatory factors, pro-
and molecules, whose main task is to distinguish between           duction of enzymes, receptors and immunoglobulins, cell
self and non-self. This is an active process, which leads to       differentiation and proliferation. In metabolic terms, this
tolerance of own cells of the body and protection from             means varying activity in synthesis of both structural
invading pathogens. The basic state of immune activity             and export proteins. Therefore determination of in vivo
shifts rapidly following recognition of harmful antigens           protein synthesis in immune-competent cells enables an
or molecules, which initiates immune responses aimed at            assessment of their functional activity and may lead to a



Key words: cell subpopulation, flow cytometry, l-[2 H5 ]phenylalanine, mass spectrometry, protein synthesis, tonsil.
Abbreviations: APE, atom percentage excess; DC, dendritic cell; FSR, fractional protein synthesis rate; MNC, mononuclear cell;
MTBSTFA, N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide; SSA, 5-sulphosalicylic acid dihydrate; SRBC, sheep red blood
cell; UPPP, uvulopalatopharyngoplasty.
Correspondence: Dr Anna Januszkiewicz (email anna.januszkiewicz@karolinska.se).


                                                                                                              C   2005 The Biochemical Society
180   A. Januszkiewicz and others


      greater understanding of the balance between health and                   Table 1 Demographic data of subjects
      disease.                                                                  studied
         We have demonstrated previously [1] that the in vivo                   Values are medians (range). BMI, body mass index.
      rate of protein synthesis decreases in circulating peri-
                                                                                Variable                           Subjects
      pheral blood T-lymphocytes in healthy volunteers in
      response to a 6 h infusion of stress hormones as a model                  Sex (female/male)                  6/6
      for surgical stress. The decrease is even more pronounced                 Age (years)                        46 (24–60)
      after intravenous exposure to endotoxin, serving as a hu-                 Weight (kg)                        75 (56–90)
      man model of the initial phase of the acute response to                   Height (m)                         1.71 (1.50–1.90)
      sepsis [2]. In contrast, a 6 h infusion of cortisol alone does            BMI (kg/m2 )                       25 (19–29)
      not change protein synthesis in circulating T-lympho-
      cytes of healthy volunteers either immediately or 18 h
      after the infusion period [3].                                   in sterile water together with unlabelled phenylalanine
         However, under normal conditions, circulating lym-            (Ajinomoto Company, Tokyo, Japan) to a concentration
      phocytes represent only 2 % of the total lymphocyte              of 20 g/l and an appropriate isotopic enrichment. The
      pool in the body and therefore they may not reflect alter-        solutions were prepared, heat-sterilized and stored in
      ations in the whole lymphoid tissue [4]. The palatine ton-       sterile containers.
      sils belong to the MALT (mucosa-associated lymphoid
      tissue) and play an important role in protecting both the        Subjects and experimental protocol
      gastrointestinal and upper respiratory tract from invading       Twelve healthy patients, scheduled for elective ear
      antigens and molecules. They consist of specialized              surgery, UPPP (uvulopalatopharyngoplasty) or nose sur-
      compartments with a typical composition of immune cells          gery, were included in the present study (Table 1). The
      [5–8]. Lymphoepithelium covering the tonsillar crypts            patients were studied in the morning after an overnight
      is the place of antigen uptake by DCs (dendritic cells),         fast. In the operating room, two venous lines were inser-
      whereas, in the extrafollicular area populated mainly by         ted contralaterally into the forearm and antecubital veins
      T-lymphocytes, migrated DCs present the antigens to              and a glucose solution (Glucos Baxter 25 mg/ml buffrad;
      CD4+ T-lymphocytes, resulting in their activation, pro-          Baxter Medical AB, Kista, Sweden) was started at
      liferation and differentiation. The third compartment,           100 ml/h. An intravenous injection of l-[2 H5 ]phenyl-
      lymphoid follicles, consists predominantly of B-lympho-          alanine (45 mg/kg; 10 or 20 mole % excess) was given
      cytes which, upon activation by T-cells, proliferate and         over 10 min. Venous blood samples were taken from the
      differentiate into memory cells and immunoglobulin-              opposite arm before (time 0), and at 5, 10, 15, 30, 50, 70
      producing plasma cells. All these events are regulated           and 90 min after injection, for measurement of the iso-
      by receptor–receptor interactions as well as by cytokines        topic enrichment of phenylalanine in plasma. For the
      secreted by the antigen-presenting cells and T-lympho-           measurement of the enrichment of l-[2 H5 ]phenylalanine
      cytes [6,9]. Even in a physiological state, healthy              in the protein of T-lymphocytes, MNCs and leucocytes,
      palatine tonsils are exposed continuously to antigens and        60 ml of blood was drawn at 90 min after the start of the
      stimulated, which often is considered as a permanent             isotope injection. All patients were operated on under
      activation [10].                                                 general anaesthetic, which was induced with intravenous
         Thus the objective of the present study was to quanti-        agents and maintained with volatile anaesthetics. Within
      tatively determine in vivo protein synthesis in the human        12 min (mean; range 5–20 min) after induction of
      palatine tonsil as a reflection of the immunological activ-       anaesthesia and intubation and at 90 min after the start of
      ation under normal conditions. The metabolic activity of         the isotope injection, two tonsil biopsies were performed
      immune cells from the tonsils was compared with that             using punch forceps. The first biopsy specimen was
      of circulating peripheral cells [T-lymphocytes, total blood      frozen immediately in liquid nitrogen and then stored
      MNCs (mononuclear cells) and the whole population                at − 80 ◦ C until analysis. The second specimen was put
      of blood leucocytes]. In order to characterize the distri-       on ice and then transported for the flow cytometry
      bution of immune-competent cells in tonsil tissue versus         analysis, which was performed within 1 h. In the patients
      peripheral blood, flow cytometry analysis was per-                operated for UPPP (n = 4), the whole tonsil was obtained,
      formed.                                                          as a tonsillectomy was an initial part of the operative
                                                                       procedure. One patient was excluded due to an early
                                                                       postoperative infection.
      MATERIALS AND METHODS                                               The nature, purpose of the study and possible risks
                                                                       were explained to all patients before obtaining their
      Materials                                                        voluntary consent. The research protocol was approved
      l-[2 H5 -ring]Phenylalanine (99 atom %; Cambridge Iso-           by the Ethical Committee of the Karolinska Institutet,
      tope Laboratory, Andover, MA, U.S.A.) was dissolved              Huddinge University Hospital, Stockholm, Sweden.


      C   2005 The Biochemical Society
                                                                                            In vivo protein synthesis in human tonsil   181



Preparation of tonsil biopsies                                  the samples and standards to convert phenylalanine into
After freeze-drying the tissue, biopsies were homogen-          phenylethylamine. After incubation at 50 ◦ C overnight,
ized in 4 % (w/v) SSA (5-sulphosalicylic acid dihydrate).       100 µl of 6 M NaOH was added, samples were mixed
The homogenates were centrifuged at 16 000 g for 10 min.        and centrifuged at 16 000 g for 10 min. The supernatant
Pellets were resuspended and washed twice in 4 % (w/v)          was transferred to an Eppendorf tube and extracted with
SSA. The pellets were then dissolved in 0.3 M NaOH              500 µl of ether. The bottom layer was frozen in ethanol/
and the concentration of alkali-soluble proteins was            dry-ice and the ether layer transferred to a new tube con-
determined using a protein assay kit (Bio-Rad Labo-             taining 100 µl of 0.1 M HCl. After shaking the tubes, the
ratories, Hercules, CA, U.S.A.) with BSA as a standard.         bottom layer was frozen and the ether layer discarded.
Proteins were precipitated again and washed twice in 4 %        Samples were transferred into GC–MS vials and dried
(w/v) SSA. To the final pellet, 1 ml of 6 M HCl was added        by vacuum centrifugation and were then derivatized
and proteins were hydrolysed at 110 ◦ C for 24 h. Samples       in MTBSTFA [N-methyl-N-(t-butyldimethylsilyl)tri-
were cooled and then dried by vacuum centrifugation.            fluoroacetamide]/ethyl acetate (1/1, v/v) at 60 ◦ C for
                                                                1 h. The amount of MTBSTFA was varied depending
Separation of T-lymphocytes and MNCs                            on the sample size, which was estimated from the
Separation of T-lymphocytes was as described previously         protein concentration and dry weight. All standards were
[3]. In brief, 55 ml of blood (5 ml for MNC isolation           derivatized in 50 µl and the tonsil samples in 25–70 µl, so
and 50 ml for further separation of T-lymphocytes) was          that samples and standards gave approximately the same
decanted into six heparinized 10 ml tubes containing            peak abundance in the GC–MS analysis.
cycloheximide (0.5 mM; Sigma, St. Louis, MO, U.S.A.)               The ratio of [2 H5 ]-phenylethylamine and phenylethyl-
in order to inhibit protein synthesis. Blood was diluted        amine was determined by GC–MS (Agilent 5973n;
1/1 with PBS (pH 7.4; Unimedic, Matfors, Sweden) and            Agilent Technologies, Stockholm, Sweden). GC was
centrifuged at 800 g for 20 min at room temperature on a        performed on a HP-5MS column (30 m, 0.25 mm inner
LymphoprepTM (Nycomed Pharma AS, Oslo, Norway).                 diameter, 1 µm film; J&W Scientific, Agilent Techno-
MNCs were harvested from the interface and washed               logies). Approx. 500 ng of decarboxylated phenylalanine
three times with PBS [11]. At this step, the cell pellet from   of the standards and samples was injected. Ions m/z 180
one tube was retained for determination of protein syn-         (m + 2) and m/z 183 (m + 5) were analysed.
thesis in MNCs and was stored at − 80 ◦ C, whereas the
pellets collected from the other five tubes were processed       GC-MS analysis of plasma free
further. T-lymphocytes were separated from MNCs by              phenylalanine
the rosette method with 1 % 2-aminoethylisothiuronium           Plasma free phenylalanine was prepared and analysed by
bromide-treated SRBCs (sheep red blood cells). SRBC             GC–MS as described previously [14]. The enrichment of
rosette-forming cells were separated on a LymphoprepTM          [2 H5 ]phenylalanine in plasma was determined by moni-
[12]. SRBCs were lysed with 1 % ammonium oxalate for            toring the ions m/z 336 and 341.
5 min at room temperature. The cells were washed three
times with PBS and stored at − 80 ◦ C.                          Calculations
                                                                The enrichment of protein-bound [2 H5 ]phenylalanine
Isolation of leucocytes                                         was calculated from the ratio m + 5/m + 2 using standard
A portion (5 ml) of blood was decanted into a heparinized       curves. The protein synthesis rate was calculated as:
tube containing cycloheximide. Erythrocytes were lysed
twice with a lysing buffer consisting of 1.5 M NH4 Cl,                        FSR (%/24 h) = Ep × 100/A
0.1 M KHCO3 and 1 mM Na-EDTA (pH 7.5) for 10 min
                                                                where FSR is the fractional protein synthesis rate, Ep
in 37 ◦ C. The cells were washed with PBS and stored at
                                                                is the protein-bound enrichment of [2 H5 ]phenylalanine
− 80 ◦ C.
                                                                (APE), A is the area under the curve for [2 H5 ]phenyl-
                                                                alanine enrichment (APE) in plasma over time (in days).
GC–MS analysis of protein-bound
phenylalanine                                                   Phenotypic characterization of cells
Samples were prepared for GC–MS analysis largely as             The isolated blood leucocytes and cell suspensions of ton-
described previously [13]. The dried protein hydrolysates       sil tissue were washed in PBS supplemented with 0.5 %
were dissolved in 400 µl of 0.5 M sodium citrate buffer         BSA in polystyrene round-bottom tubes and then the
(pH 6.3) and were filtered through a 0.22 µm centrifugal         cell surface was stained. Antibodies used for phenotypic
filter device. Phenylalanine standards containing 0–0.25         analysis were combinations of directly conjugated anti-
APE (atom percentage excess) of l-[2 H5 ]phenylalanine          CD45, anti-CD4, anti-CD8, anti-CD3, anti-CD16,
were dissolved in 200 µl of citrate buffer. A suspension        anti-CD19, anti-HLA-DR (Becton Dickinson, San
(100 µl) of l-tyrosine decarboxylase (0.7 units plus            Jose, CA, U.S.A.), anti-CD14, anti-CD15, anti-CD56,
0.25 mg of pyridoxal phosphate) was added to 200 µl of          anti-CD25 and anti-CD54 (DakoCytomation, Glostrup,


                                                                                                    C   2005 The Biochemical Society
182   A. Januszkiewicz and others


      Table 2 FSRs in the circulating immune cells of peripheral                 Table 3 Cell-surface markers determined by flow cyto-
      blood (T-lymphocytes, MNCs and leucocytes) and in the                      metric phenotyping in tonsil tissue and blood in healthy
      tonsils of healthy subjects (n = 11)                                       subjects (n = 11)
                                                                                 Values are means (range). ∗ P < 0.01 compared with blood cells. All markers,
                        FSR (%/24 h)
                                                                                 expressed as a percentage, were calculated from the combined lymphocyte and
      Subject no.       T-lymphocytes     MNCs         Leucocytes   Tonsil       monocyte gate, except for CD15+ cells in blood (†), which were also calculated
                                                                                 from the granulocyte gate.
       1                 6.2               9.3         1.5          22.0
       2                14.1              14.5         3.3          22.6         Cell-surface marker                Tonsil                        Blood
       3                 5.4               9.9         1.3          23.2         CD3+                               62   (44–81)                  65   (57–75)
       4                 8.0               6.6         3.2          24.9         CD3+ /CD4+                         53   (34–75)∗                 42   (28–54)
       5                15.0              13.4         3.0          17.1         CD3+ /CD8+                          9   (6–13)∗                  20   (14–26)
       6                11.1               7.6         2.9          29.4         CD19+                              30   (16–49)∗                  8   (5–12)
       7                11.0              10.2         4.5          26.7         CD14+                               0   (0–2)∗                   14   (7–20)
       8                11.2              11.0         4.8          22.5         CD15+                               1   (0–4)                     0   (0–1)
       9                10.5               9.9         3.0          28.8         CD15+                               1   (0–4)∗                   99   (94–99)†
      10                15.8              15.7         5.1           8.9         CD16+                               3   (1–10)∗                  27   (15–37)
      11                 9.6              10.6         2.9          24.2         CD56+                               2   (1–9)∗                   18   (7–40)
      Mean + S.D.
           −            10.7 + 3.4
                             −            10.8 + 2.8
                                               −       3.2 + 1.2
                                                           −        22.8 + 5.7
                                                                         −       HLA-DR+                            35   (18–56)                  26   (20–33)
                                                                                 CD25+                               2   (1–3)                     1   (0–2)
                                                                                 CD54+                               7   (4–14)∗                  13   (8–19)
      Denmark) sera. The cells were incubated for 15 min at
      4 ◦ C and then washed and analysed immediately. Non-
      specific antigen binding was examined by staining the                       and monocyte gate. CD15+ cells in blood were also cal-
      cells with fluorochrome-labelled isotype-matched nor-                       culated from the granulocyte gate. CD3+ T-lymphocytes
      mal mouse immunoglobulins. The cells were analysed                         were the most abundant cell populations in both the
      by a FACSCalibur flow cytometer (Becton Dickinson).                         tonsils and blood. The ratio of CD4+ T-lymphocytes
      Data were evaluated by Cell Quest software (Becton                         to CD3+ T-lymphocytes was higher among tonsillar
      Dickinson).                                                                cells compared with the peripheral blood cells (P < 0.01).
                                                                                 A higher proportion of CD19+ B-lymphocytes was
      Statistical analysis                                                       observed in the tonsil as compared to blood (P < 0.01). In
      Data are presented as means + S.D., or medians (ranges)
                                  −                                              contrast, CD3+ /CD8+ (P < 0.01) and CD14+ monocytes
      when appropriate. Comparative statistics of CD markers                     (P < 0.01), CD15+ granulocytes (P < 0.01), CD16+
      between the tonsillar and blood cells were performed                       monocyte subpopulation and natural killer lymphocytes
      using the non-parametric Wilcoxon rank sum test. Corre-                    (P < 0.01) and CD56+ natural killer lymphocytes (P <
      lation between the protein synthesis rate and the ex-                      0.01) were present at low levels in tonsils compared with
      pression of CD19+ B-lymphocytes in the tonsils was                         blood. CD19+ B-lymphocytes did not correlate with the
      tested by Spearman’s test.                                                 FSR in the tonsils (r = 0.53, P = 0.09).
                                                                                    HLA-DR+ was expressed in 30 % of the cells and no
                                                                                 differences in the distribution between blood and tonsils
      RESULTS                                                                    were observed. Another activity marker, CD25+ , was
      In vivo protein synthesis in blood and tonsils in healthy                  detectable at very low levels in both the tonsillar and
      adult patients was determined quantitatively during a                      circulating blood cells. The expression of the adhesion
      90 min period by intravenously injecting l-[2 H5 ]phenyl-                  molecule CD54 was higher in blood than in the tonsil
      alanine and then measuring its enrichment in immune-                       (P < 0.01).
      competent cells. The in vivo FSR in the palatine tonsils
      was 22.8 + 5.7 %/24 h (Table 2). This protein synthesis
                −                                                                DISCUSSION
      rate was found to be higher compared with peripheral
      blood. In peripheral blood cells, the in vivo FSR                          For the first time, the present study has quantified in vivo
      was 3.2 + 1.2 %/24 h in the whole leucocyte population
              −                                                                  protein synthesis in human lymphoid tissue represented
      (Table 2). In total MNCs and sorted T-lymphocytes                          by the palatine tonsil. The rate of protein synthesis in the
      from peripheral blood, the FSRs were 10.8 + 2.8 and
                                                     −                           tonsil was twice as high as that in the circulating T-lym-
      10.7 + 3.4 %/24 h respectively (Table 2).
           −                                                                     phocytes and MNCs. CD3+ lymphocytes were the most
         The phenotypic analysis of the tonsillar and blood                      abundant cell population in the tonsil.
      cell subsets by flow cytometry is shown in Table 3. All                       Tonsil biopsy is a well-documented technique, which
      markers were calculated from the combined lymphocyte                       enables access to the lymphoreticular system relatively


      C    2005 The Biochemical Society
                                                                                             In vivo protein synthesis in human tonsil   183



easily even on an outpatient basis [15]. In our present         lower compared with the lymphoid tissue, still indicates
study, all biopsies were performed in anaesthetized             a high metabolic activity of these cells, even in the resting
patients after induction of anaesthesia, but before the start   state. This can be explained by the fact that T-lympho-
of the operation. The biopsies were taken with punch            cytes, as well as monocytes, play an important immuno-
forceps from the upper part of the tonsil (except for four      regulatory role, including production of receptors, cyto-
patients scheduled for UPPP, where tonsillectomy was a          kines and other regulatory factors. The low FSR of
part of the surgical procedure) and no complications due        approx. 3 %/24 h seen in the total population of leuco-
to the bleeding were observed. The time from induction of       cytes in the peripheral blood is also in agreement with
anaesthesia until tonsil biopsies was 12 min (mean; range       an earlier study in healthy volunteers [2]. This probably
5–20 min). As the total incorporation time was 90 min,          reflects the fact that neutrophils, which are the most
70–85 min of the incorporation period was in the awake          abundant cell population in peripheral blood, are not
state before induction of anaesthesia. Separate biopsies        active in the absence of stimulation [23].
were taken for determination of protein synthesis and              In order to characterize cell populations in the tonsil
for phenotypic characterization. All biopsies used for          and to compare them with the populations of peripheral
the latter purpose contained tonsillar tissue. Although the     blood, flow cytometry was performed. Our results
palatine tonsils consist of three different compartments        showed that immune-competent cells were distributed
with specific anatomy and functions, immunologically             differently in the lymphoid organs and blood, which is in
active cells are present in all three compartments. The         accordance with a previous report [24]. CD3+ T-lympho-
relatively low scatter of the in vivo protein synthesis rate    cytes were the dominant cell population, present
in the palatine tonsil, being of similar magnitude to that      in the same proportions in both pools investigated. In
of the circulating cells, suggests that the biopsy specimens    contrast with blood, the percentage of B-lymphocytes
were representative for the tonsil tissue.                      was three times higher in the palatine tonsil, as shown
   The in vivo FSR in human palatine tonsil was 23 %/           previously [25]. It is known that lymphoid organs are the
24 h, which is a relatively high value, probably reflect-        location where B-cells mature and differentiate and the
ing the continuous process of antigen scanning, which           question can be raised whether the high metabolic activity
involves production of regulatory factors and cell inter-       observed in tonsils may be related to the activity of B-
actions, as well as cellular activation and proliferation.      lymphocytes rather than T-lymphocytes. However, there
This protein turnover is in the same range as that seen in      was no correlation between the protein synthesis rate in
the human liver, known for its high metabolic activity,         the tonsil and the expression of CD19+ B-lymphocytes
which has an FSR of total (both stationary and export)          in the tonsillar cells.
proteins of approx. 25 %/24 h [16]. In the human intes-            The expression of HLA-DR+ and CD25+ is often used
tinal mucosa, a twice as high FSR is reported, mainly           to characterize the state of activation of immune cells.
due to a rapid cell turnover [17,18]. Also, in rats a simi-     Despite the higher protein synthesis in tonsillar tissue,
lar pattern to that demonstrated in human subjects is           HLA-DR+ (MHC class II), which is up-regulated on
reported with a lower FSR in the peripheral blood MNCs          monocytes, DCs and B-cells during antigen presentation
compared with lymphoid organs, such as bone marrow,             as well as expressed on a subpopulation of activated
thymus and spleen [19,20].                                      T-cells, was equally distributed between blood and ton-
   It can be speculated that the high protein turnover          sils. Similarly, CD25+ , up-regulated transiently mainly
seen in the tonsil reflects a more pronounced cell pro-          on activated T-lymphocytes [26] and expressed on very
liferation taking place in lymphoid organs. In vitro stu-       few cells, showed no difference in the distribution bet-
dies have shown an increased thymidine and uridine              ween the tonsil and blood.
incorporation in the tonsillar cells compared with the             In summary, the present study has shown that the
blood lymphocytes [21]. On the other hand, the prolifer-        in vivo protein synthesis rate in the human palatine ton-
ation rates in lymph nodes are not higher than pro-             sils is higher compared with circulating peripheral blood
liferation rates in the T-lymphocyte subsets in an ex vivo      cells. This high protein turnover may reflect the conti-
study using the Ki67 antigen as a proliferation marker          nuous state of immunological activation seen in the
[22]. As there are no data available on the in vivo pro-        human tonsil. Nevertheless, the differences seen in
liferation rates in human tonsils, the high protein syn-        the rates of protein synthesis between the tonsils and
thesis rate observed in our study still may be related to       blood cannot be explained by activity markers and sub-
an enhanced proliferation of immune-competent cells.            populations in immune-competent cells.
   In vivo protein synthesis of the circulating peripheral
blood cells was determined in parallel with that of the
tonsils. Both T-lymphocytes and MNCs had a similar              ACKNOWLEDGMENTS
in vivo FSR of approx. 11 %/24 h, which is consistent
with our previous studies performed in healthy volun-                                                           a
                                                                We would like to thank Viveka Gustavsson, Hern´ n
teers [1–3]. The protein synthesis rate, although 50 %          Concha Quezada, Charlotte Tammik, Christina Hebert,


                                                                                                     C   2005 The Biochemical Society
184   A. Januszkiewicz and others


                                  ¨
      Eva Skoog and Inga Hellstrom for the excellent technical        13 Slater, C., Preston, T., McMillan, D. C., Falconer, J. S.
      assistance. We also thank Gareth Morgan for interesting            and Fearon, K. C. H. (1995) GC/MS analysis of
                                                                         [2 H5 ]phenylalanine at very low enrichment: measurement
      discussions on tonsil histology. A. J. was a recipient of          of protein synthesis in health and disease.
      the ESPEN (European Society for Clinical Nutrition and             J. Mass Spectrom. 30, 1325–1332
      Metabolism) Research Fellowship 1998. The study was                                         e
                                                                      14 McNurlan, M. A., Ess´ n, P., Thorell, A. et al. (1994)
                                                                         Response of protein synthesis in human skeletal muscle
      supported by grants from the Swedish Medical Research              to insulin: an investigation with l-[2 H5 ]phenylalanine.
      Council (04210 and 14244).                                         Am. J. Physiol. 267, E102–E108
                                                                      15 Faust, R. A., Henry, K., Dailey, P. et al. (1996) Outpatient
                                                                         biopsies of the palatine tonsil: access to lymphoid tissue for
                                                                         assessment of human immunodeficiency virus RNA titers.
      REFERENCES                                                         Otolaryngol. Head Neck Surg. 114, 593–598
                                                                                                      e
                                                                      16 Barle, H., Nyberg, B., Ess´ n, P. et al. (1997) The synthesis
       1 Januszkiewicz, A., Ess´ n, P., McNurlan, M. A.,
                                 e                                       rates of total liver protein and plasma albumin determined
         Ringd´ n, O., Garlick, P. J. and Wernerman, J. (2001) A
                e                                                        simultaneously in vivo in humans. Hepatology 25,
         combined stress hormone infusion decreases in vivo              154–158
         protein synthesis in human T lymphocytes in healthy          17 Charlton, M., Ahlman, B. and Nair, K. S. (2000) The effect
         volunteers. Metab., Clin. Exp. 50, 1308–1314                    of insulin on human small intestinal mucosal protein
       2 Januszkiewicz, A., Lor´ , K., Ess´ n, P. et al. (2002)
                                 e         e                             synthesis. Gastroenterology 118, 299–306
         Response of in vivo protein synthesis in T lymphocytes       18 Nakshabendi, I. M., McKee, R., Downie, S., Russell, R. I.
         and leucocytes to an endotoxin challenge in healthy             and Rennie, M. J. (1999) Rates of small intestinal mucosal
         volunteers. Clin. Exp. Immunol. 130, 263–270                    protein synthesis in human jejunum and ileum.
       3 Januszkiewicz, A., Ess´ n, P., McNurlan, M. A.,
                                 e                                       Am. J. Physiol. 277, E1028–E1031
         Ringd´ n, O., Wernerman, J. and Garlick, P. J. (2000)
                e                                                     19 Papet, I., Ruot, B., Breuille, D. et al. (2002) Bacterial
         In vivo protein synthesis of circulating human T                infection affects protein synthesis in primary lymphoid
         lymphocytes does not respond to a cortisol challenge            tissues and circulating lymphocytes of rats. J. Nutr. 132,
         within 24 h. Acta Anaesthesiol. Scand. 44, 202–209              2028–2032
       4 Westermann, J. and Pabst, R. (1990) Lymphocyte subsets in    20 Papet, I., Dardevet, D., Sornet, C. et al. (2003) Acute phase
         the blood: a diagnostic window on the lymphoid system?          protein levels and thymus, spleen and plasma protein
         Immunol. Today 11, 406–410                                      synthesis rates differ in adult and old rats. J. Nutr. 133,
       5 Nave, H., Gebert, A. and Pabst, R. (2001) Morphology and        215–219
         immunology of the human palatine tonsil. Anat. Embryol.      21 Siegel, G. (1979) Biochemical characterization of the
         204, 367–373                                                    proliferation pool of lymphatic cells in the human tonsil
       6 van Kempen, M. J., Rijkers, G. T. and Van Cauwenberge,          and demonstration of the age-dependent pool size
         P. B. (2000) The immune response in adenoids and tonsils.       reduction. Acta Otolaryngol. 87, 560–566
         Int. Arch. Allergy Immunol. 122, 8–19                        22 Fleury, S., Rizzardi, G. P., Chapuis, A. et al. (2000)
       7 Perry, M. and Whyte, A. (1998) Immunology of the tonsils.       Long-term kinetics of T cell production in HIV-infected
         Immunol. Today 19, 414–421                                      subjects treated with highly active antiretroviral therapy.
       8 Brandtzaeg, P. (2003) Immunology of tonsils and adenoids:       Proc. Natl. Acad. Sci. U.S.A. 97, 5393–5398
         everything the ENT surgeon needs to know. Int. J.            23 Fletcher, J., Haynes, A. P. and Crouch, S. M. (1990)
         Pediatr. Otorhinolaryngol. 67 (Suppl. 1), S69–S76               Acquired abnormalities of polymorphonuclear neutrophil
                                        ¨
       9 Andersson, J., Abrams, J., Bjork, L. et al. (1994)              function. Blood Rev. 4, 103–110
         Concomitant in vivo production of 19 different cytokines     24 Rosenmann, E., Rabinowitz, R. and Schlesinger, M. (1998)
         in human tonsils. Immunology 83, 16–24                          Lymphocyte subsets in human tonsils: the effect of age and
      10 Surjan, Jr, L. (1987) Tonsils and lympho-epithelial             infection. Pediatr. Allergy Immunol. 9, 161–167
         structures in the pharynx as immuno-barriers.                          e
                                                                      25 Ringd´ n, O. (1976) Activation of human lymphocyte
         Acta Otolaryngol. 103, 369–372                                  subpopulations by rabbit anti-human β 2 -microglobulin
      11 Boyum, A. (1976) Isolation of lymphocytes, granulocytes         and by lipopolysaccharide. Scand. J. Immunol. 5, 891–900
         and macrophages. Scand. J. Immunol. Suppl. 5, 9–15           26 Brandtzaeg, P. and Halstensen, T. S. (1992) Immunology
      12 Parker, J. W. (1979) Immunologic basis for the redefinition      and immunopathology of tonsils. Adv. Otorhinolaryngol.
         of malignant lymphomas. Am. J. Clin. Pathol. 72, 670–686        47, 64–75


                                                         Received 9 September 2004/26 October 2004; accepted 9 November 2004
                                                         Published as Immediate Publication 9 November 2004, DOI 10.1042/CS20040271




      C   2005 The Biochemical Society

				
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