Connective Tissue Stains Cells

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					                                   CONNECTIVE TISSUE
                                     CELLS & STAINS



Specific Learning Objectives

1. List the specialized stains used to visualize specific extracellular matrix components.

2. Know the structure and function of the resident cells of connective tissue: fibroblasts,
   macrophages, plasma cells, mast cells and adipocytes.

3. Identify the transient cells of connective tissue: lymphocytes, neutrophils, eosinophils and
    basophils.


I. Introduction
     Connective tissue consists of a diverse group of tissues that provide support and connections
     among the other tissues of the body. Connective tissue forms a continuous compartment
     throughout the body, limited by the basement membranes of epithelia, muscle, nerves and
     vascular endothelium. All connective tissues are derived from mesenchyme, the embryonic
     connective tissue.

   The components of connective tissues are cells suspended in an extracellular matrix.
   Structurally, connective tissues contain few cell-cell contacts, in sharp contrast to the
   epithelial tissues discussed previously. Relatively few cells exist per unit area and they are
   dispersed in a large amount of extracellular matrix. This matrix contains fibers and a ground
   substance composed of proteoglycans, glycoproteins and water. Connective tissue is a large
   category including connective tissue proper (loose, dense, regular, irregular), adipose, blood,
   hematopoetic tissue, lymphatic tissue, cartilage and bone. The proportions and types of
   fibers, ground substance, cells, and fluid content vary considerably among these classes of
   connective tissue, and reflect the diverse functions of this broad group.

II. Extracellular Matrix (ECM)
    A. Fibers
       1. Collagen – visualize with Mallory Trichrome stain. Colors connective tissue collagen
          blue. Collagens provide tensile strength to tissues.
       2. Reticular – visualize with silver stain. Colors fine collagen III reticular fibers black.
          Reticular fibers provide a fine mesh of support to solid organs, such as the liver,
          pancreas, lymph nodes, spleen, and even patches of adipose tissue.
       3. Elastic – visualize with Verhoeff’s (black) or orcein (reddish-brown). Elastic fibers
          provide for stretch and recoil in ligaments, large arteries, and the epiglottis.
    B. Ground substance
       1. Proteoglycans – visualize with Periodic Acid Schiff’s (PAS); a stain which colors
          carbohydrates bright pink. Proteoglycans absorb water to hydrate and increase the
          rigidity of connective tissues.


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       2. Glycoproteins – These occur in less density than other connective tissue elements,
          therefore each specific glycoprotein requires a tagged antibody to visualize its
          location. Glycoproteins tend to have specialized functions in connective tissues such
          as anchors and signaling molecules.
       3. Water – All connective tissue are characterized by a high degree of water content.
          This is usually reflected in tissue specimens as clear spaces between other elements.
       4. Matrix Metalloproteinases (MMPs) - This group of zinc dependant enzymes
          degrades all the components of the extracellular matrix (ECM). These enzymes are
          secreted as inactive precursors and held in check by inhibitors, (the tissue inhibitors of
          metalloproteinases, TIMPs). The MMPs include collagenases, stromelysins (degrade
          basement membrane), elastases and gelatinases (degrade collagen I). Connective
          tissue matrix is degraded during the development, growth and turnover of most
          organs. Even such a routine process as ovulation requires MMPs to thin the ovarian
          wall prior to release of the egg.


III. Connective Tissue Cells

   The cells of connective tissue are generally classified in two categories: A) resident cells
   which are regularly present in the connective tissue, and B) transient cells that migrate here
   from the blood.

   A. Resident Cells

       1. Fibroblasts
           Fibroblasts are spindle shaped cells that produce the fibers and ground substance of
           ordinary connective tissue. The secretory process is not polarized; product is
           released at all cell surfaces. Fibroblasts display a slightly basophilic cytoplasm
           (staining rRNA) and a nucleus with a prominent nucleolus. Taken together, both
           indicate active protein synthesis. With H & E stained sections, however, the
           cytoplasmic extensions are often difficult to discern. But one should be able to
           appreciate that the large pink staining collagen bundles of the extracellular matrix are
           outside the cell membrane, rather than within the fibroblast cytoplasm. Fibroblasts
           tend to anchor themselves to extracellular collagen fibers.


       2. Macrophages
           Macrophages are large phagocytic cells common in loose connective tissue. They
           are also found in hematopoetic tissues, lungs, liver and the spleen. Macrophages are
           approximately 20 µm in diameter (2.5-3 X the diameter of a RBC), with an irregular,
           oval shape. In the light microscope, macrophages may appear to have a “foamy”
           cytoplasm, but are most easily recognized after phagocytosis of vital dyes, such as
           trypan blue or carbon. In the electron microscope, macrophages exhibit the classic
           organelles of a phagocytic cell: 1) irregular, highly folded surface (phagocytic
           pseudopodia); 2) numerous cytoplasmic vesicles, heterogeneous in size and density
           (primary lysosomes, phagosomes, secondary lysosomes, residual bodies); 3) small



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   pinocytic vesicles (clathrin coated and smooth endosomes); and 4) a euchromatic
   nucleus.

   Macrophages phagocytose aging ECM elements and cells, infectious
   microorganisms, and cellular debris from acute inflammatory reactions after
   infection or trauma. For example, in the spleen, macrophages dispose of aging red
   blood cells. The second major function of macrophages is to process ingested
   antigens and to present them to lymphocytes in order to activate the immune
   response. Macrophages exhibit the Major HistoCompatibility class II (MHC II)
   surface antigens found on all antigen presenter cells. But macrophages are also
   secretory cells that release their products both by constitutive and regulated
   secretion. Macrophages secrete a broad battery of proteins related to the immune
   response, inflammation and hematopoietic growth regulation. The two primary
   cytokine secretions of macrophages are interleukin 1, an activator of helper T cells,
   and tumor necrosis factor , an inflammatory mediator. Macrophages are part of a
   complex network of immune cells.

   Macrophages are part of the Mononuclear Phagocyte System. All the cells of this
   group are derived from stem cells in the bone marrow that differentiate into
   monocytes, which circulate in the blood, and further differentiate into macrophages
   in the peripheral tissues. In different organs macrophages have separate names:
               Connective Tissue = macrophages or histiocyes
               Liver = Kupffer cells
               Lung = alveolar macrophages or dust cells
               Nervous System = microglia
               Bone = osteoclasts
               Skin = Langerhans cells



3. Plasma Cells
    Plasma cells differentiate from B-lymphocytes to produce antibodies directed against
    one specific antigen. Plasma cells are commonly seen in the respiratory and
    gastrointestinal tracts, salivary glands and lymph nodes. The electron microscopic
    structure of a plasma cell is an excellent study of structure/function correlation. The
    nucleus contains large heterochromatin clumps (“clockface” chromatin) and a
    distinct nucleolus. Together, these indicate that very little of the genome is actively
    transcribing, but yet synthesis of rRNA is brisk. The cytoplasm is primarily filled
    with rough endoplasmic reticulum, typical of a cell very active in protein synthesis.
    Finally, there are no storage vesicles in the cytoplasm; therefore all the synthesized
    protein must be released by constitutive secretion. In the light microscope, a plasma
    cell has a basophilic cytoplasm, due to the large quantities of RNA associated rough
    endoplasmic reticulum, a clear cytoplasmic zone near the nucleus, which is the site
    of the Golgi, and a “clockface” nucleus.




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4. Mast Cells
    Mast cells are large (20 µm) ovoid cells often found beside small blood vessels.
    They are easily identified in areolar (loose connective tissue) spreads, but difficult to
    find in tissue sections. Mast cells migrate from the blood to fully differentiate in the
    connective tissue compartment. The hallmark of a mast cell is a cytoplasm packed
    with uniform secretory granules. The content of the granules is so condensed that
    binding stains are visualized metachromaticly; i.e. tightly packed dye molecules shift
    the wavelength of light which they reflect (toluidine blue stains reddish-purple). In
    the electron microscope, there is some variability to the fixed appearance of the
    granules: mast cells of the lung have scroll and lamellar appearing granules; mast
    cells of the skin, blood vessels, GI tract and joint synovium have very densely
    staining homogenous granules.

   Fc receptors on the mast cell become primed with IgE molecules secreted by plasma
   cells at the initial exposure to an antigen. Upon the second expose to the same
   antigen, mast cells can now immediately bind the antigen to the primed IgE on their
   surface. This triggers an intracellular cascade of calcium, resulting in a rapid (1-5
   minutes), extensive release or “degranulation” of their stored granules. Mast cell
   granules store preformed, or primary mediators (heparin, histamine, acid hydrolases,
   neutral proteases and chemotactic factors for eosinophils and neutrophils). At the
   same time, mast cell activation triggers the synthesis of lipid mediators of
   inflammation.      Within 5-30 minutes, the enzyme cyclooxygenase produces
   prostaglandin D2, which stimulates bronchial smooth muscle contraction; while the
   enzyme lipoxygenase produces leukotriene C4, a vasodilator. A third level of mast
   cell activation is the actual activation of protein synthesis. The cytokines tumor
   necrosis factor , interleukins, platelet activating factor and GM-CSF (granulocyte
   monocyte- colony stimulating factor) are produced hours after mast cell IgE binding.
   Together these primary and secondary mediators contribute to the immediate
   hypersensitivity reactions, allergic responses such as hives, asthma and hay fever,
   and anaphylaxis.

   a. Heparin, a sulfated proteoglycan, acts as an anticoagulant. Also, the negative
       charges on the molecule are capable of forming ionic bonds with other granular
       constituents and may aid in condensing and transporting the storage granules
       within the mast cell cytoplasm.
   b. Histamine, formed from the decarboxylation of histidine, has contrary effects on
       arterioles; it contracts visceral arterial & bronchial smooth muscle, and relaxes
       peripheral arterial smooth muscle. This diverges blood flow from the organs to
       the peripheral tissues. Histamine also increases vascular permeability. Immune
       cells can more easily exit the blood, and plasma fluid leaks out into the
       connective tissue compartment.
   c. Slow-reacting substance of anaphylaxis (leukotriene C4), stimulates contraction of
       venule endothelial cells, slightly separating their lateral borders. Fluid and
       plasma proteins pass from the blood and into the interstitial fluid compartment
       creating edema. White blood cells also egress from the plasma into the
       connective tissue space. The process of white blood cell migration through
       endothelial gaps is called diapedesis.


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   d. Cytokines - chemotactic factors for eosinophils, monocytes and neutrophils attract
       white blood cells to the site.

   Mast cells also interact with the autonomic nervous system. They respond to
   substance P and to acetylcholine by degranulating and by synthesizing newly formed
   lipid mediators and cytokines. Mast cells, especially in the intestinal tract, receive
   input from and provide signals to the nervous system.


5. Adipocytes
    Adipocytes are dynamically involved in the storage and metabolism of energy. Lipids
    are the highest caloric food per weight, and are therefore the most efficient means to
    store energy. Besides, there are only limited stores of glucose (as glycogen) in
    skeletal muscle and liver, and no significant stores of proteins short of degrading
    muscle mass. Insulin stimulates adipocyte uptake of glucose and its conversion into
    triglycerides. Insulin binding also increases adipocyte synthesis and secretion of
    lipoprotein lipase (LPL). LPL is bound by endothelial cells lining local blood
    vessels where it enzymatically attacks lipids of lipoproteins circulating in the blood.
    Therefore lipoprotein fatty acids are liberated in adipose tissue and are also taken up
    for storage. Adipocyte fat stores are mobilized by the binding of epinephrine,
    norepinephrine, glucagon or ACTH to a specific receptor. Intracellular cAMP
    increases and activates intracellular lipase (via phosphorylation), which degrades
    lipids and releases free fatty acids into capillaries. Within the blood, free fatty acids
    are carried by albumin.

   Adipocytes can be identified in histological sections as very large, round cells,
   containing one large vacuole. This vacuole contained lipid, but it has been lost
   during the tissue processing necessary for light microscopy. The accumulation of a
   single lipid inclusion flattens and laterally displaces the nucleus, and creates a thin
   rim of cytoplasm. This unilocular fat cell is the predominant cell of white adipose
   tissue (white fat). Special staining procedures such as Sudan IV (red) and osmium
   tetraoxide (black) can be used to visualize fat ladened, intact adipocytes.

   A second type of adipose cell is called a multilocular or brown adipocyte. The
   adipocytes of brown fat contain multiple lipid droplets of varying size. There is also
   more cytoplasm in these cells and the nucleus is not pushed to the periphery of the
   cell. The brown tissue color is due to the high concentration of cytochromes in its
   abundant mitochondria and an extensive capillary bed. A unique protein,
   thermogenin, uncouples electron transport from oxidative phosphorylation, resulting
   in lower production of ATP, with more energy released as heat. Multilocular
   adipose tissue functions in thermoregulation and is prominent in hibernating species
   and in newborn primates, where it is located in the axillae, kidney fat pad and in the
   posterior triangles of the neck.




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B. Transient Cells
    Transient cells migrate into the connective tissue from the blood in response to specific
    stimuli.
    1. Lymphocytes
        Lymphocytes are small cells (6-8 µm), with a large heterochomatic nucleus.
        Lymphocytes are actually two classes of defensive cells: T-lymphocytes - long lived,
        involved in cell mediated immunity, and B-lymphocytes - involved in production of
        plasma cells and antibody. Lymphocytes can be found in normal blood and are
        abundant in the connective tissue of the respiratory tract and the gastrointestinal tract
        where they provide protection against foreign antigens.


   2,3,4. Eosinophils, Neutrophils and Basophils
       These three cell types migrate from the blood in response to immune stimuli or tissue
       injury. These cells are the granulocytes of the white blood cells, as all three store
       secretory material in their cytoplasm. They are named for their staining
       characteristics in the light microscope. Specific details on their functions will be
       covered in the Immunology and Hematology courses.




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posted:4/17/2011
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