Connective Tissue Cells
Laboratory 6

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Required Reading: Ross and Romrell: Chapter 5, pp. 106-125 and Chapter 6, pp. 126-131

Gartner and Hiatt: Chapter 3, pp. 42-59

YOUR JOB DURING THIS LAB: You are responsible for identifying each of the connective tissue cells listed below at both the light and electron microscopic level. You should also be able to summarize their important function(s) briefly. Since this is a lot of work, I have included here a synopsis of the more detailed cell descriptions to be found in your textbook. You are, however, responsible for the material in the textbook.

CT cells may be classified as being one of two general types; indigenous (fixed) and immigrant (wandering). The indigenous cells are a stable population responsible for the synthesis and maintenance of the extracellular matrix. The immigrant cells are usually involved in short-term events such as defense.

INDIGENOUS CELLS

Primitive mesenchymal cell

This cell originates in the embryonic mesenchyme. As the embryo grows and its cells differentiate, multipotent undifferentiated CT cells decrease in relative numbers until in the adult CT they persist as single cells, usually lying alongside of small blood vessels. In the LM they are pale, with an elongated nucleus and ill-defined cytoplasm. They are very inconspicuous and cannot be identified with any certainty in the lab. In the adult these cells may be adipose cell precursors, but this is debated by some investigators. The mesenchymal cells may also be involved in assisting the development of new capillaries.

Fibroblast

This is the predominant cell type of the CT proper. Fibroblasts are responsible for the production of most of the extracellular matrix components and will be found everywhere. They arise from primitive mesenchymal cells in the embryo. With the LM they are oval, with a flattened nucleus, and spindle-shaped or stellate cytoplasm. Usually, only the nucleus may be clearly distinguished. With the EM, fibroblasts may be seen to have abundant ribosome-studded endoplasmic reticulum and a large Golgi complex, reflecting the intense biosynthetic activity of these cells.

Adipose Cell

Adipose, or fat, cells also derive originally from the primitive mesenchymal cells. After differentiation, adipose cells are spherical and large (up to 120 um in diameter). The bulk of the cell volume consists of a fat droplet which under the LM appears as a hole surrounded by a thin rim of cytoplasm. If the plane of section happens to pass through the single, peripheral nucleus, the adipose cell will look like a "signet ring". Larger groupings of adipose cells may appear like "chicken-wire". Adipose cells do not divide, nor do they move. Their plasma membrane contains receptors for insulin, glucagon, other hormones and sympathetic neurotransmitters. Adipose cells function as a dynamic store of neutral triglycerides, which after synthesis from imported fatty acids and glucose remain in the cell for an average of 4 days. The balance between deposit and withdrawal of fat is regulated by the autonomic nervous system.

Brown fat, as contrasted with the white fat just discussed, is found in newborn humans and particularly in hibernating animals. Here, specialized adipose cells contain multiple small fat droplets and numerous mitochondria. Cytochromes in the mitochondria lend the characteristic color to brown fat. Brown fat cells are capable of rapidly metabolizing their own lipid stores to produce heat.

Mast Cell

Mast cells also originate from primitive mesenchymal cells and in adults may often be found near small blood vessels. They do divide to produce new mast cells. In the LM, mast cell cytoplasm is usually oval and filled with granules. The granules are neutral with H & E stain, but stain deep purple with thiazin dyes such as toluidine blue (metachromasia). There is a central nucleus with condensed chromatin. In the EM, the cell surface reveals irregular microvilli. The cytoplasmic granules are seen to be membrane-bounded and filled with dense, homogeneous material.

Mast cells move freely but slowly through the CT. They do not phagocytose significant amounts of materials. They function to stimulate and influence other CT cells in various ways via the release of mast cell granules. These granules contain heparin-protein complex, histamine, phospholipid, anaphylactic factors, and basic proteins including proteolytic enzymes and phosphatases. Mast cells will degranulate and release granules either individually or in groups in response to a variety of stimuli including: a) mechanical trauma, such as wounding or pinching, b) radiant energy such as x-rays, UV light or heat, c) chemical agents such as detergents, bacterial toxins, snake venoms. Perhaps of the greatest importance, molecules from certain parasites and invertebrates and antigens when combined with IgE antibodies on the mast cell surface will stimulate degranulation.

The effects of mast cell degranulation and histamine release are many. Histamine causes blood venules to leak, which yields edema, makes smooth muscles contract, and causes itching. Another mast cell factor stimulates fibroblasts to proliferate and synthesize more extracellular matrix. A secreted mast cell peptide will attract eosinophils into the CT and stimulate bone marrow production of eosinophils. Mast cells also produce a fibrinolysin which inhibits local blood clotting in order to allow more rapid ingress of cells into the CT for healing and defensive purposes. Mast cells are long-lived and do not commit suicide during degranulation.


IMMIGRANT CELLS

Neutrophil - Polymorphonuclear leucocyte

"Polys" originate in the bone marrow and enter the CT from the bloodstream. They tend to stick to capillary walls (they marginate) where about half of the neutrophil population may be found at any one time. Marginated neutrophils may migrate between endothelial cells into the connective tissue; this process is termed diapedesis.

Neutrophils in the LM are round cells, about 9um in diameter with a multilobed nucleus and indistinct colorless granules when observed with H & E. The EM allows resolution of large dense primary granules (lysosomes) and smaller, less dense secondary or "specific" granules. Both granule types are synthesized in immature cells and stored for later use. Polys contain abundant glycogen supplies as energy sources in an anaerobic environment. They have substantial amounts of actin and myosin filaments in their cytoplasm providing a mechanism for cell motility and for phagocytosis.

Neutrophils are highly motile in the CT. They may speed along at up to 4mm per day. They respond chemotactically to fragments of serum complement, bacterial substances or lymphokines released by lymphocytes. They do not divide and survive for less than one day. Neutrophils are also kamikazes; they sacrifice themselves to save you. Upon phagocytosis neutrophils die.

Neutrophils function as a rapid defense system via phagocytosis. They recognize bacteria which have been coated by serum complement or antibody molecules (IgG), and surround the bacterium with cytoplasmic extensions which fuse to internalize the invader in a membrane-lined phagosome. Then, the two types of granules sequentially fuse with the phagosome membrane, releasing their contents to kill the enclosed bacterium. Bacteria are killed in a complex process which involves low pH, antimicrobial substances (lysozyme and others), superoxide radicals, proteases, lipases, and carbohydrases. Some types of bacteria somehow inhibit granule fusion and survive within the neutrophils; these include mycobacterium tuberculosis and toxoplasma gondii.

Monocyte

Monocytes originate in the bone marrow and enter the CT via the blood stream. With the LM, they have a kidney-shaped nucleus, abundant cytoplasm and are about 10-12 um in diameter. They contain usually inconspicuous azurophilic granules. They are motile and respond to the same chemotactic factors as do neutrophils. They marginate and enter the CT more slowly than do neutrophils, with monocytes moving 1-2 mm per day. They may phagocytose, but high phagocytic activity occurs only after monocytes differentiate into macrophages.


Macrophage

Macrophages differentiate from monocytes. In the LM, they appear as cells with a very extensive and irregular cytoplasm perhaps containing a variety of particles and vacuoles. The nucleus is small and irregular with a visible nucleolus. The EM reveals that macrophage cytoplasm contains small membrane-limited vesicles filled with lysosomal enzymes and larger granules containing the remnants of phagocytosed material.

Macrophage have three major functions: 1) Defense, by the ingestion and killing of bacteria and fungi which have been coated by IgG or serum complement; 2) Garbage collection, by ingestion of damaged and senile cells, dead bacteria, other particles; and, 3) Storage of wastes in residual bodies. Such wastes include carbons and tars and are particularly notable in the macrophages present in the lungs of smokers. Macrophages also secrete a great variety of products including collagenase, lysozyme, interferon and others. They are very dynamic cells and seem to be involved in almost all aspects of CT defense in some way or other.

There exist different populations of macrophage. These include: alveolar macrophages in the lung, peritoneal macrophages in the peritoneal cavity, synovial macrophages in the synovial cavity, Kupffer cells in the liver, and histiocytes in the CT proper.


Lymphocyte

These originate in the bone marrow or from lymphatic nodules in the CT itself. They are small cells about 8 um in diameter with a round nucleus and scanty cytoplasm. They actively migrate thru the CT and when doing so, may exhibit a deformed nucleus. Lymphocytes display cell surface antibodies which act as potential receptors for antigens. The cells move thru the CT looking for their particular antigen. When this antigen is found, they respond actively in a complicated process to be discussed later in the course (lymphoid tissue lectures). For some lymphocytes (B cells), antigenic stimulation eventually results in differentiation to plasma cells.


Plasma Cell

As stated, these arise from B lymphocyte differentiation after antigenic triggering. They are round or oval cells with a strongly basophilic cytoplasm and eccentric round nucleus. They show clumped chromatin and a central nucleolus ("wagon-wheel"). With the EM, the cytoplasm is seen to be filled with RER, consistent with the sole function of these cells as an antibody factory. Plasma cells are non-motile, non-phagocytic, and when fully formed, unable to divide.

They function to synthesize and secrete enormous amounts of immunoglobulin (antibody) molecules; up to 2000 molecules per second.


Eosinophil

These arise in the bone marrow and enter the CT from the blood. They are about 9 um in diameter, have a bilobed nucleus, and a cytoplasm filled with refractile eosinophilic granules. The EM shows that these granules are membrane-bound and contain a dense crystalloid in an amorphous matrix. Eosinophilic granules, therefore, may be distinguished morphologically from those of other cell types.

Eosinophils are motile in response to chemotactic factors from mast cells or parasites and in response to the presence of antigen-antibody complexes. They may be somewhat phagocytic and cannot divide. In the CT they live about 8 days. The actual function of eosinophils is still obscure, but they are found in large numbers in instances of parasitic diseases.


LABORATORY EXERCISE

Cell Types Found in Connective Tissue

YOUR JOB HERE: You should review the ten or so different types of cells you might expect to find in connective tissue. Certain of these cells will be found preferentially in certain types of CT, or in certain regions of the body, or at certain times, depending for example upon the local immune state of the organism. You will be held responsible for identifying each of these cells types at the light and electron microscopic level. You should understand the basic function(s) of each cell type and be prepared to describe this in one or two sentences.

Images to introduce the connective tissue cell types.

C14: Three MAST CELLS are seen on this slide with darkly staining histamine granules. Label (a) indicates an elastic fiber which is just barely visible; (b) indicates a FIBROBLAST nucleus. Note the relative abundance of fibroblasts, compared to mast cells. When in doubt, if looking at small dark nuclei in CT, call them fibroblast nuclei (you will usually be right).

C15: The large red oval cell is a rat mast cell. Note the larger appearance when compared with the other cells noted; in part, this is due to the cytoplasmic staining of the mast cells. (As an aside, the red "donuts" you see on this slide are the nuclei of rat eosinophils, which differ in nuclear morphology from human eosinophils.) EOSINOPHILS are immigrant blood cells which invade the CT, usually when the body is faced with parasitic stress.

C16: This electron micrograph is of a mast cell from the intestinal lamina propria. The granule content is obvious. The neighboring two cells are LYMPHOCYTES.

C17: PLASMA CELLS are seen here as the largest cells with a dark, "cart-wheel" heterochromatin pattern in the eccentric nucleus, with highly basophilic cytoplasm. (a) indicates a large lymphocyte, as defined by the lack of an extensive cytoplasmic skirt. The smaller cells are lymphocytes and some plasma cells. Lymphocytes will have a dark, "smudged" nucleus and very little cytoplasm in relation to nuclear volume.

C18: An EM of a plasma cell, indicating that the extreme basophilia seen in the preceding slide is caused by an abundance of rough endoplasmic reticulum producing antibody molecules for export.

C19: This slide is a low magnification view of the air sac region in the lung. The living tissue had been infiltrated with a dark marker substance, which was then phagocytized ("eaten") by MACROPHAGES. Here you can see the macrophages clustered around a pulmonary vein. The lung is one of the body areas particularly rich in macrophages.

C20: This is a high resolution plastic-section light micrograph of an area similar to that shown in C19. A macrophage is visible directly in the air space (this is outside world!) and is identified by its cytoplasm filled with various inclusions. The other cells on this slide compose the alveolar walls of lung air sac and will be discussed later in the course. Question: how can the macrophage survive in the air-sac outside space? How do you think it gets there? Why would it want to?

C21: Here you see ADIPOSE cells (adipocytes), exhibiting their characteristic "chicken-wire" look. The large empty spaces represent the cytoplasmic areas originally filled with lipid, extracted during slide preparation. Only a very attenuated rim of cytoplasm is present with the nuclei squeezed between the lipid and the internal face of the plasma membrane. The arrows indicate some of the adipocyte nuclei. Some capillaries are also visible; adipose tissue is well-vascularized.

 

Suggested glass slides to observe connective tissue cell types.

Slide 5 - adipose tissue: Here the lipids have been retained and stained by fixation in osmium tetroxide. Those empty spaces you saw in image C21 are now filled with material. Note also the interspersed dense, irregular CT which remains unstained. The collagen bundles are visible as wavy branches.

Slide 8 - mesentery whole mount: Almost all of the cells here are fibroblasts. Why do some nuclei look much darker and narrower than the others?

Slide 9 - mesentery whole mount: The large granular cells are mast cells.

Slide 114 - stomach: This is a good representative slide to study the cells of both dense irregular CT (in the sub-mucosa) and in the loose CT of the lamina propria (just below the epithelium). Higher power would be helpful in looking at the lamina propria.

Slides 115 (fundic stomach) and 120 (jejunum): Look here between the epithelium and the muscle layers in the dense CT to find fibroblasts, mast cells, lymphocytes and even the occasional plasma cell, macrophage, eosinophil, etc.

Slide 197 - mammary gland: Ignoring for now the ducts and the secretory areas, concentrate on the neighboring CT regions. Here you should find lots of adipose cells, as well as some plasma cells and mast cells.

Continue practicing your identification of connective tissue cells by looking at other slides in your glass slide collection. Any slide of the gastrointestinal tract, most slides of the respiratory tract, and many others of particular organs will be helpful in this regard. The identification of connective tissue cells is one of the few times where 100X oil immersion is usually advisable, but remember not to start your search at this level of magnification.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Image C14
Mast cells


Image C15
Mesentary whole mount


Image C16
TEM of mast cell and lymphocytes


Image C17
Plasma cells


Image C18
TEM of plasma cell


Image C19
Pulmonary macrophages


Image C20
Pulmonary macrophage


Image C21
Unilocular adipose cells