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Connective Tissue Learning objectives * Understand the feature and classification of the . * Understand the structure and function of varied composition of the . * Know the composition of the matrix. * Know the features of fibers. * Know the composition of the . * Know the basic structure and function of the dense connective tissue, reticular tissue and . General characteristic:

- Connective tissue is formed by cells and (ECM). - It differ from the . - It has a small number of cells and a large amount of extracellular matrix. - The cells in C. T have no polarity. . That means they have no the free surface and the basal surface. - They are scattered throughout the ECM. - The extracellular matrix is composed of . fibers ( constitute the formed elements) , an . amorphous ground substance and . tissue fluid. - Connective tissue originate from the , which is embryonal C. T. . The cells have multiple developmental potentialities. . They have the bility to differentiate different kinds of C. T cells, endothelial cells and smooth muscle cells. - Connective tissue forms a vast and continuous compartment throughout the body, bounded by the basal laminae of the various epithelia and by the basal or external laminae of muscle cells and nerve-supporting cells. - Different types of connective tissue are responsible for a variety of functions. Functions of connective tissues:

The functions of the various connective tissues are reflected in the types of cells and fibers present within the tissue and the composition of the ground substance in the ECM.

- Binding and packing of tissue ……….CT Proper; - Connect, anchor and support………... and ; - Transport of metabolites……………..Through ground substance; - Defense against infection…………….Lymphocytes, ; - Repair of injury………………………. tissue; - Fat storage…………………………… Adipose tissue. Loose connective tissue Classification of connective tissues * Is based on the composition and organization of its cellular and extracellular components and on its functions. - Embryonic connective tissue . Mesenchyme . Mucous connective tissue - Connective tissue proper . Loose connective tissue, cellular C.T - Reticular - Adipose tissue - Pigmental. C.T - Lymphatic.C.T . Dense connective tissue - Regular - Irregular - Specialized connective tissue . Сkeletal C.T - - . Fluid connective T - Blood . Hemopoietic tissue Embryonic connective tissue a= mesenchymal tissue, b= Mucous connective tissue (Wharton’s jelly) Mesenchymal connective tissue. Fetal pig. Paraffin section. ×540. Mesenchymal connective tissue of the fetus is very immature and cellular. The mesenchymal cells (MeC) are stellate-shaped to fusiform cells, whose cytoplasm (c) can be distinguished from the surrounding matrix. The nuclei (N) are pale and centrally located. The ground substance is semifluid rich in hyaluronan (), but with very little in consistency and contains slender reticular fibers. The vascularity of this tissue is evidenced by the presence of blood vessels (BV). Mesenchyme is the embryological tissue from which all types of supporting/connective tissue are derived. Mesenchymal cells are relatively unspecialised and are capable of differentiation into all supporting tissue types. Some mesenchymal cells remain in mature supporting tissue and act as stem cells (see Ch. 2). Mesenchymal cells have an irregular, star (stellate) or spindle (fusiform) shape, with delicate branching cytoplasmic extensions which form an interlacing network throughout the tissue. The nuclei have dispersed chromatin and visible nucleoli. The matrix consists almost exclusively of blue- ground substance without mature fibres, facilitating diffusion of metabolites to and from developing tissues. Electron micrograph (EM) of two apposed mesenchymal cells in the tendon of a fetus. The nucleus of one cell is mainly euchromatic; that of the adjacent cell has relatively more heterochromatin, which reflects a different functional state. Profiles of rough endoplasmic reticulum (RER) and a prominent Golgi complex (GC) occupy the cytoplasm of one cell; mitochondria (Mi), in the other. Points of membrane contact between cells (circles), more common in developing tissue, are uncommon in adult connective tissue. 14,000×. Mucoid C,T

Mucous tissue of umbilical cord named “Wharton’s jelly”. H&E stain. Low magnification. Mucous connective tissue. Umbilical cord. Human. Paraffin section. ×132. This example of mucous connective tissue (Wharton’s jelly) was derived from the umbilical cord of a fetus. Observe the obvious differences between the two embryonic tissues. The matrix of mesenchymal connective tissue contains no collagenous fibers, whereas this connective tissue displays a loose network of haphazardly arranged fibers (CF). The cells are no longer mesenchymal cells; instead, they are (F), although morphologically they resemble each other. The empty looking spaces (arrows) are areas where the ground substance was extracted during specimen preparation. Inset. . Umbilical cord. Human. Paraffin section. ×270. Note the centrally placed nucleus (N) and the fusiform shape of the cytoplasm (c) of this fibroblast. Mucoid C,T Connective Tissue * Connective tissue proper - Loose connective tissue . Cellular connective T . Reticular T . Adipose T . Pigmental C.T . Lymphatic. C.T - Dense connective tissue . Regular . Irregular Connective tissue proper Loose connective tissue (Areolar C.T)

- Characterized by loosely arranged fibers and abundant cells of various types. - The ground substance, is abundant; in fact, it occupies more volume than the fibers do. -It has a viscous to gel-like consistency and plays an important role in the diffusion of oxygen and nutrients from the small vessels, as well as in the diffusion of carbon dioxide and metabolic wastes back to the vessels. Areolar Connective Tissue * Description - Gel-like matrix with: . all three fiber types (collagen, reticular, elastic) for support . Ground substance is made up by also made and screted by the fibroblasts. - Cells – fibroblasts, macrophages, mast cells, white blood cells * Function - Wraps and cushions organs - Holds and conveys tissue fluid - Important role in inflammation Main battlefield in fight against infection * Defenders gather at infection sites - Macrophages - Plasma cells - Mast cells - Neutrophils, lymphocytes, and eosinophils Loose Connective Tissue Lamina propria of trachea trachea. Human· H.E. stain Loose Connective Tissue Subcutaneous connective tissue spread. Rat· AFLGOG stain Areolar Connective Tissue

* Location - Widely distributed under epithelia - Packages organs - Surrounds capillaries - Form endoneurium ------endomysium Loose (areolar) connective tissue. Paraffin section. ×132. This photomicrograph depicts a whole mount of mesentery, through its entire thickness. The two large mast cells (MC) are easily identified, since they are the largest cells in the field and possess a granular cytoplasm. Although their cytoplasms are not visible, it is still possible to recognize two other cell types due to their nuclear morphology. Fibroblasts (F) possess oval nuclei that are paler and larger than the nuclei of macrophages (M). The semifluid ground substance (GS) through which tissue fluid percolates is invisible, since it was extracted during the preparation of the tissues. However, two types of fibers, the thicker, wavy, ribbon-like, interlacing collagen fibers (CF) and the thin, straight, branching elastic fibers (EF), are well demonstrated. A spread, whole-mount preparation of loose (areolar) connective tissue (H&E plus an stain, 200×). The most prominent features are the many black, thin, branched elastin fibers; some are straight; others, which have been cut, are wavy. The collagen fibers are the thick pink (eosinophilic) cables that appear to be in the background. There are many nuclei in this field of view; most are fibroblasts, some may be macrophages, but it is difficult to identify any of the cells with certainty. Areolar connective tissue wraps and cushions organs A section of inactive (H&E, 125×). The dark-staining nuclei of the inactive mammary epithelial cells are seen clustered as acini and ducts. The epithelial cells are surrounded by a very sparse, somewhat cellular (mostly fibroblasts) bed of loose connective tissue. Also in the field of view are several blood vessels, areas of adipose cell-rich loose connective tissue, and some regions of dense irregular connective tissue. A section of showing the region just below the (H&E, 250×). The loose connective tissue that is just under the epidermis is called the papillary . It is filled with delicate fibers, blood vessels, and often the nerve fibers that innervate the epidermis. The lower part of the field in this image shows some of the collagen fibers that are part of the dense regular connective tissue of the reticular dermis that lies under the papillary dermis. A section of the tips of two intestinal villi from the jejunum (H&E, 320×). The epithelium is simple columnar and is made up of intestinal absorptive cells and mucus-secreting goblet cells. The central part of each villus is filled with loose connective tissue; in this location, the term lamina propria is given to this connective tissue in accordance with the nomenclature used for hollow organs. It is difficult to identify most of the individual cells, but several can be identified eosinophils, fibroblasts, a neutrophil, and several large cells that could be macrophages. The lamina propria also has blood and lymphatic capillaries and several isolated smooth muscle cells that are extensions of another structural feature of hollow organs the muscularis mucosae. Loose connective tissue, Lymphatic connective. T lamina propria of colon, Mallory, ×250 Cellular connective tissue in the of just below the epithelium (H&E, 100×). The connective tissue here is highly cellular (mostly fibroblasts) and filled with many small- diameter collagen fibers. Many lymphatic vessels and a few blood vessels are interspersed throughout the connective tissue. Cellular connective tissue in the stroma of and Cellular connective tissue in the stroma of ovary Pigment connective tissue (Iris Van Gieson) Reticular Connective Tissue

* Description - Network of reticular fibers in loose ground substance * Function - Form a soft, internal – supports other cell types * Location - lymphoid organs . Lymph nodes, and . - - Adrenal gland

Reticular tissue

(a) The diagram shows only the fibers and attached reticular cells (free, transient cells are not represented). Reticular fibers of type III collagen are produced and enveloped by the reticular cells, forming an elaborate network through which interstitial fluid or lymph and wandering cells from blood pass continuously. (b) The micrograph shows a silver-stained section of in which reticular fibers are seen as irregular black lines. Reticular cells are also heavily stained and dark. Most of the smaller, more lightly stained cells are lymphocytes passing through the lymph node. X200. Silver. A silver stain of the cortex of a fetal adrenal gland (140×). There are many thin to very thin black fibers in this image; some are in the capsule, some divide clusters of adrenal parenchymal cells into columnar groups, and the finest are associated with cell clusters in the lower third of the image. In these silver-stained sections of adrenal cortex (a) and lymph node (b), networks of delicate, black reticular fibers are prominent. These fibers serve as a supportive stroma in most lymphoid and hematopoietic organs and many endocrine glands. The fibers consist of type III collagen that is heavily glycosylated, producing the black argyrophilia. Cell nuclei are also dark, but cytoplasm is unstained. X100. Reticular .C.T fibers forming the internal skeleton of the spleen Reticular Fibers Human liver. Silver impregnation Adipose Tissue * Description - Closely packed - Have nucleus pushed to one side by fat droplet. * Function - Provides reserve food fuel - Insulates against heat loss - Supports and protects organs * Location - Under skin - Around kidneys - Behind eyeballs, within abdomen and in breasts White or unilocular adipose tissue is commonly seen in sections of many human organs. (a) Large white adipocytes (A) are seen in the connective tissue associated with small blood vessels. The fat cells are empty because lipid was dissolved away in slide preparation. Nuclei at the cell membranes are visible in some of the fat cells. X100. H&E. (b) Large (empty) adipocytes predominate in this typical , which shows only a small portion of microvasculature. In a single histologic section, nuclei of most very large adipocytes are not included. X100. H&E. (c) Tissue was fixed here with osmium tetroxide, which preserves lipid (L) and stains it black. Many adipocytes in this slide retain at least part of their large lipid droplets. X440. Osmium tetroxide. (d) The specimen here was from a young mammal, and the adipocytes marked with asterisks are not yet unilocular, having many small lipid droplets in their cytoplasm, which indicates that their differentiation is not yet complete. The eccentric nuclei of unilocular cells are indicated by arrowheads. X200. PT. Adipose CT Connective tissue proper; Dense Connective Tissue

Irregular and Regular

Dense irregular connective tissue: * Description: - mostly collagen fibers, Some elastic fibers . Typically, the fibers are arranged in bundles oriented in various directions (thus, the term irregular). - Cells are sparse and are typically of a single type, the fibroblast. - Relatively little ground Substance. •Function: - Withstands tension - Provides significant structural strength •Location: - Dermis of skin - of digestive tract - Fibrous capsules of joints and organs Dense Irregular CT = dermis Section of skin showing the epidermis and dermis (Mallory’s trichrome stain, 100×). The lower three-quarters of the field is the dense irregular connective tissue of the reticular dermis, which in this specimen demonstrates the intermingled nature of the blue collagen fibers and reddish- brown elastin fibers particularly well. Note that the elastin fibers closer to the papillary dermis are thinner than those in the lower part of the field. The loose connective tissue of the papillary dermis has very little elastin, but some short, thin fibers can be seen. The basal layer of the epidermis is highly pigmented owing to the presence of melanin granules. Dense Irregular Connective Tissue Eyelid Stain: azan; magnification: × 70

Dense Irregular Connective Tissue Renal Capsule Scanning electron microscopy; magnification: × 5000 A section of the submucosa of the intestine (H&E, 250×). The dense irregular arrangement of the collagen fibers is evident; note the significant space between the fibers. A section of the submucosa of the intestine Masson’s trichrome stain. The dense irregular arrangement of the collagen fibers is evident; note the significant space between the fibers. A different field of view (H&E, 125×) of the same section of inactive mammary gland. The irregularly arranged collagen fibers are closely packed and have many more fibroblasts per unit area. On the right side of the field are a few groups of mammary epithelial cells surrounded by loose connective tissue. Electron micrograph of dense irregular connective tissue. Parts of four fibroblasts are seen in this micrograph. The nuclei of all four have large amounts of euchromatin; the cell nucleus in the lower right center has a prominent nucleolus. The cytoplasm of all four cells (and the cytoplasmic processes below them) is nearly completely filled with rER and other organelles; note that many of the rER cisternae are dilated. All these cellular features are indicative of a high rate of protein (mostly collagen) synthesis and secretion. The extracellular space is filled with many bundles of collagen fibers the cross-banded structure of which can be seen. (Courtesy of Jeffrey B. Kerr.) Dense Connective Tissue regular connective tissue: * Description: - The fibers are arranged in parallel array and are densely packed to provide maximum strength, and some elastic fibers - The Fibroblasts that produce and maintain the fibers are packed and aligned between fiber bundles. - little ECM * Function: - Attaches muscle to bone - Attaches bone to bone - Withstands great stress in one direction * Location: - Is the main functional component of and , - Aponeuroses , cornea (special regular C.T) - around muscles------Tendons are cordlike structures that attach muscle to bone. ( van Gieson ) Dense Regular Connective Tissue Tendon Stain: alum hematoxylin-eosin; magnification: × 240

1 Peritendineum internum Stain: alum hematoxylin-eosin; magnification: × 130

1 Fibril bundles 2 Nuclei of tendon cells 3 Cytoplasmic processes of tendon cells 4 Cytoplasm of tendon cells with rER 5 Lysosomes Electron microscopy; magnification: × 8300 (a) Micrograph shows a longitudinal section of dense regular connective tissue in a tendon. Long, parallel bundles of collagen fibers fill the spaces between the elongated nuclei of . X100. H&E stain. (b) The electron micrograph shows one in a cross section of tendon, revealing that the sparse cytoplasm of the fibrocytes is divided into numerous thin cytoplasmic processes extending among adjacent collagen fibers. X25,000. Dense regular connective tissue tendon Electron micrograph of sections of tendon. CLINICAL POINT - Tendinitis inflammation of tendon or its sheath is caused mostly by overuse injury after repetitive motion from recreational, athletic, or occupational activities. - Because of a poor blood supply, tendon injuries are often hard to treat, especially in older people. . Most common are tendinitis of wrist (tenosynovitis), Achilles tendon, patella (jumper’s knee), lateral (tennis elbow) or medial (golfer’s elbow) epicondyles, and rotator cuff (swimmer’s shoulder). - Tendinitis may also be linked to inflammatory disease (e.g., rheumatoid arthritis, gout). . Treatment options include physical rehabilitation, cortisone injection, and use of NSAIDs or cyclooxygenase-2 inhibitors. . Promising novel approaches are use of nitric oxide delivery patches, shock-wave therapy, stem cell injections, and gene transfer strategies. . Surgery is reserved for patients with severe tendon damage who are unlikely to respond to other treatments. Dense Regular CT = ligament join bone to bone Parallel bundles of collagen (pink) with elastin fibers (ribbon candy) and non-aligned fibroblasts.

The fibers of are arranged in multiple layers (orthogonal array) of the cornea Electron micrograph of the cornea (35,000×). Note the uniform diameter of all the collagen fibers, the absence of any fibroblasts or their cell processes, and the orthogonal arrangement of the nearly uniform lamellae of fiber bundles. (Courtesy of Don W. Fawcett and M. Jakus.) Aponeuroses (white fibrous tissue that takes the place of a tendon in flat muscles having a wide area of attachment ) resemble broad, flattened tendons. Instead of fibers lying in parallel arrays, the fibers of aponeuroses are arranged in multiple layers (orthogonal array). Elastic Fibers Corium

Auricle ( Flap) Elastic lamellae Aorta

1 Elastic lamellae 2 Collagen fibrils 3 Smooth muscle cells Electron microscopy; magnification: × 4500

Elastic Fibers Corium

Extracellular matrix (ECM) Extracellular matrix (ECM) Description:

* The extracellular matrix (ECM) is a complex and intricate structural network that surrounds and supports cells within the connective tissue. - It contains a variety of fibers such as . Collagen and . Elastic fibers that are formed from different types of structural proteins. - and ground substance; . ; - aggrecan, syndecan; . Multiadhesive glycoproteins; - , ); and . (GAG); - , keratan sulfate, hyaluronan. * Extracellular matrix (ECM) functions: - ECM provides mechanical and structural support of cells within the connective tissue. - It influences extracellular communication, - It is biochemical barrier. - It regulating metabolic functions of the cells surrounded by the matrix. - It anchors cells within tissues through cell-to-ECM adhesion molecules, - It provides pathways for (e.g., during wound repair). - It exerts a regulatory effect on and cell differentiation. - It is capable of binding and retaining growth factors, which in turn modulate cell growth. - It influences the transmission of information across the plasma membrane of the connective tissue cells. - ECM is a dynamic and interactive system that informs cells about the biochemical and mechanical changes in their extracellular environment. Connective tissue fibers

Collagen Fiber * Description: - Acidophilic, thick, birefrigent - Very resistant to traction . extensibility …..5% . very high tensile strength - Most abundant proteins of the body . 20% of total body proteins - High in glycine, alanine, proline, hydroxyproline, hydroxylysine, poor in sulfur, not Tryptophan. Collagen fibers

Subunits of , the most abundant collagen, assemble to form extremely strong fibrils, which are then bundled together further by other into much larger structures called collagen fibers. (b) The large bundles of type I collagen fibrils (C) appear as acidophilic collagen fibers in connective tissues, where they may fill the extracellular space. Subunits for these fibers were secreted by the fibroblasts (arrows) associated with them. X400. H&E. Collagen fibers: SEM & TEM EM of collagen fibrils beside a fibroblast. The distinctive axial periodicity of type I collagen is shown when the fibrils are sectioned in the longitudinal plane (upper left). Repeating dark and light segments every 67 nm along the length of each fibril are due to a staggered arrangement of constituent tropocollagen molecules. The cytoplasm of the adjacent fibroblast contains multiple, closely packed RER cisternae. The RER consists of flattened membranous sacs (*) studded with ribosomes and organized as parallel stacks. A mitochondrion (Mi) lies in the intervening cytoplasm. 50,000×. (Courtesy of Dr. L. Arsenault)

EM of collagen fibrils in transverse section. In the extracellular matrix (ECM), collagen fibrils appear as circular profiles with relatively uniform diameters. The process of a fibroblast in this area contains profiles of RER and other organelles in its cytoplasm. 50,000×. Electron micrograph of collagen showing the distinctive 68-nm cross-banded pattern of the collagen fibrils and associated proteins. (Courtesyof Jeffery B. Kerr.) Collagen fibrils in dense irregular connective tissue.

Note the random orientation of collagen fibrils that overlie and crisscross each other in the connective tissue matrix. 65,000. Shown here are the relationships among type I collagen molecules, fibrils, fibers, and bundles. 1. Rodlike triple-helix collagen molecules, each 300 nm long, self-assemble in a highly organized, lengthwise arrangement of overlapping regions. 2. The regular, overlapping arrangement of subunits continues as large collagen fibrils are assembled. 3. This structure causes fibrils to have characteristic cross striations with alternating dark and light bands when observed in the EM. 4. Fibrils assemble further and are linked together in larger collagen fibers visible by light microscopy. 5. Type I fibers often form into still larger aggregates bundled and linked together by other collagens. The photo shows an SEM view of type I collagen fibrils closely aggregated as part of a collagen fiber. Striations are visible on the surface of the fibrils.

Diagram showing the molecular character of a type 1 collagen fibril in increasing order of structure. COLLAGEN Several classes of collagens are identified on the basis of their polymerization pattern.

- Most of the collagen molecules polymerize into supramolecular aggregates such as fibrils or networks, and they are divided into several subgroups on the basis of their structural or amino acid sequence similarities. • Fibrillar collagens include types I, II, III, V, and XI collagen molecules. - These types are aggregate to form 68-nm-banded fibrils . • Fibril-associated collagens with interrupted triple helixes (FACITs) have interruptions in their triple helixes that provide flexibility to the molecule. - They are located on the surface of different fibrils and are represented by types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII collagens. . Type IX collagen molecule binds and interacts with type II collagen in the cartilage at the intersections of the fibrils. - It serves to stabilize this tissue by binding type II collagen fibrils with proteoglycans of the ECM. • Hexagonal open network–forming collagens are represented by; - collagen types VIII (Descemet's membrane of cornea) and - X (associated with cartilage fibrils). • Transmembrane collagens are represented by; - Types XIII (in the focal adhesions), - XVII (in the hemidesmosomes), - XXIII (in metastatic cells), and - XXV (a brain-specific collagen). • Multiplexins (collagens with multiple triple-helix domains and interruptions) comprise collagen types XV and XVIII, which reside in the . • Basement membrane forming collagens include type IV collagen ( the collagen suprastructure in the basement membrane of epithelial cells

- Type VI collagen, which forms beaded filaments, that attaches the chondrocytes to the matrix, covalently bound to type I collagen fibrils -Type VII collagen, which forms that attach the basement membrane to the ECM. - Recently identified collagen types (XXVI to XXIX) have not been fully characterized. . Collagen types Types of Collagen, Composition, Location, and Function

Types of Collagen, Composition, Location, and Function

Collagen fibrils often consist of more than one type of collagen. - Usually different types of fibrillar collagens assemble into fibrils composed of more than one type of collagen molecule. For example, . Type I collagen fibrils often contain small amounts of types II, III, V, and XI. . assembly of type I collagen fibrils is proceeded by formation of a fibrillar core containing type V and type XI molecules. . In addition, small amounts of type II and III collagen molecules are incorporated into type I collagen fibrils. - Collagen types V and XI are important regulators of fibrillogenesis. - Fully mature collagen fibers are usually associated with the FACIT family of collagen molecules that reside on their surfaces. For example: . Type I fibrils are associated with type XII and type XIV collagens. . Collagen type IX resides on the surface of the type II fibril and anchors it to proteoglycans and other components of the cartilaginous ECM . Collagen fibril

Collagen biosynthesis: from the cytoplasm to the Matrix Collagen biosynthesis Cleavage of the procollagen molecule.

Cleavage of the procollagen molecule. Illustration showing the procollagen molecule with N and C termini. Scissors in the top illustration show where C and N terminals are cleaved by carboxy and aminopeptidase, respectively, from the procollagen molecule to form the collagen molecule. On the C terminus of the molecule, the sugar subunit is GlcNac (N- acetylglucosamine) attached to mannose (Man)n. Globular N-terminal propeptide is smaller and has short triple-helical and nontriple-helical domains, whereas C- terminal propeptide is larger with a single nontriple helical domain. Collagen molecules are synthesized by various types of connective tissue and epithelial cells.

- Collagen molecules are largely synthesized by connective tissue cells include: . Fibroblasts, in cartilage, in bone, pericytes in blood vessels, and . the collagen molecules of basement membrane are produced by epithelial cells. - The synthesis of collagen is regulated by complex interactions among growth factors, and hormones, . transforming growth factor (TGF-) and . platelet-derived growth factor (PDGF) stimulate collagen synthesis by fibroblasts, whereas . steroid hormones (glucocorticoids) inhibit its synthesis. Collagen fibers are degraded either by proteolytic or phagocytic pathways.

- All proteins in the body are being continually degraded and resynthesized. . These processes allow tissues to grow and to undergo remodeling. . Further degradation is continued by specific enzymes called proteinases.

- Excessive collagen degradation is observed in several diseases (e.g., degradation of cartilage collagen in rheumatoid arthritis or bone collagen in osteoporosis).

- collagen molecules are degraded mainly by two different pathways: • Proteolytic degradation occurs outside the cells through the activity of enzymes called matrix metalloproteinases (MMPs). - These enzymes are synthesized and secreted into the ECM by a variety of connective tissue cells (fibroblasts, chondrocytes, monocytes, neutrophils, and macrophages), some epithelial cells (keratinocytes in the epidermis), and cancer cells. - The MMPs include: . collagenases (which degrade type I, II, III, and X collagens); . gelatinases (which degrade most types of denatured collagens, laminin, fibronectin, and elastin); . stromelysins (which degrade proteoglycans, fibronectin, and denatured collagens); . matrilysins (which degrade type IV collagen and proteoglycans); . membrane-type MMPs (which are produced by cancer cells and have a potent pericellular fibrinolytic activity); and . metalloelastases (which degrade elastin, type IV collagen, and laminin). - In general, triple-helical undenatured forms of collagen molecules are resistant to\ degradation by MMPs. - Damaged or denatured collagen (gelatin) is degraded by many MMPs, with gelatinases playing the prominent role. . MMP activity can be specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs). . Because MMPs are secreted by invasive (migrating) cancer cells, researchers are investigating synthetic therapeutic agents that inhibit the activity of MMPs to control the spread of cancer cells.

• Phagocytotic degradation occurs intracellularly and involves macrophages to remove components of the ECM. - Fibroblasts are also capable of phagocytosing and degrading collagen fibrils within the lysosomes of the cell. Clinical points * Keloid Formation - The body responds to wounds, including those caused by surgical intervention, by forming that repair the damage first with weak type III collagen that is later replaced by type I collagen, which is much stronger. - Some individuals, especially African Americans, form an overabundance of collagen in the healing process, thus developing elevated scars called keloids. . The collagen fibers in keloids are much larger, more eosinophilic said to have a “glassy” appearance than the normal, fibrillar, collagen. . Moreover, keloids are hypocellular, although they frequently display clusters of fibroblasts distributed among the large, glassy collagen fiber bundles. * Scurvy - Scurvy, a condition characterized by bleeding gums and loose teeth among other symptoms, results from a vitamin C deficiency. . Vitamin C is necessary for hydroxylation of proline for proper tropocollagen formation giving rise to fibrils necessary for maintaining teeth in their bony sockets.

* Ehlers - Danlos syndrome is a group of diseases in which defects in collagen type I or III result in significant structural defects in dense irregular connective tissue. - At least 10 mutations of these types of collagen are classified as Ehlers- Danlos syndrome.

* Osteogenesis imperfecta is another disease caused by a mutation in type I collagen (the α1-chain gene). Scurvy * Scleroderma (or systemic sclerosis) is a chronic, degenerative disorder that leads to overproduction of collagen as a result of an autoimmune dysfunction. - Collagen is also defective and progressively accumulates excessively throughout the body, causing clinical features that include the presence of antinuclear antibodies, widespread scarring () of the skin, and disruption of the normal architecture of internal organs such as the lungs, joints, cardiovascular system, and gastrointestinal tract. - Recent evidence suggests that upregulation of collagen gene expression in fibroblasts of affected patients is an early and critical event in its pathogenesis. * Repair with fibrosis - Following damage to cells and tissues, there is an inflammatory reaction which is responsible for eliminating the damaging agent if any, and clearing away dead tissue. - Further repair to damaged tissues is mainly delivered by support cells from connective tissues. . Briefly, there is a local proliferation of mesenchymal cells from the margins of residual normal tissue to form fibroblasts and . - These grow out to replace the area of tissue damage. - This proliferation is also associated with florid growth of new capillary blood vessels. - This combination is known as granulation tissue. - The fibroblasts and myofibroblasts secrete extracellular matrix and replace the damaged area with fibro collagenous material. - This is the basis of a collagenous scar. - Over time there is progressive remodelling to maximise the strength of the collagen and to reduce capillary vessel numbers. . This has been called healing by repair and often allows good function, but is better regarded as fibrosis and scarring as it does not restore tissue to normal. - There is evidence that epithelial cells in organs such as liver, kidney and lung can transform into a mesenchymal phenotype and contribute to fibrosis in both repair and disease. This epithelialmesenchymal transformation is subject to intense research. - Although fibroblasts are considered to be fixed cells, they are able to display some limited movement. . These cells may undergo cell division under special conditions, such as in . . when tendons are stressed because of overuse, fibroblasts may be stimulated to become chondrocytes and form cartilage matrix around themselves and transform the tendon into fibrocartilage. . Fibroblasts may differentiate into adipose cells; under pathological conditions, . fibroblasts may even differentiate into osteoblasts.

* Edema - The release of histamine and leukotrienes from mast cells during an inflammatory response elicits increased capillary permeability, resulting in an excess accumulation of and, thus, gross swelling (edema). CLINICAL POINT -Tumors of connective tissue or its mesenchymal precursors are known as . - The most common adult is malignant fibrous histiocytoma. . The cellular origin is uncertain, but immunocytochemical marker evidence indicates that it derives from perivascular mesenchymal cells. . A gene associated with this tumor, MASL1, has been identified. . a mixture of cells resembling fibroblasts, myofibroblasts, macrophages, and primitive mesenchymal cells. . Tumors typically arise in deep fascia, soft tissues of the neck or extremities, and . . Distant may spread to lung, bone, or liver. - Treatment is usually by radical resection. Reticular Fibers Reticular Fibers * Reticular fibers provide a supporting framework for the cellular constituents of various tissues and organs. - Reticular fibers and collagen type I fibers share a prominent feature. . They both consist of collagen fibrils. . Unlike collagen fibers, however, reticular fibers are composed of the individual fibrils that constitute a exhibit a 68-nm banding pattern (the same as the fibrils of type I collagen). - The fibrils have a narrow diameter (about 20 nm), exhibit a branching pattern, and typically do not bundle to form thick fibers. - Reticular fibers have a threadlike appearance. - They contain a greater relative number of sugar groups than collagen fibers (6- 12%), reticular fibers are displayed by means of the . periodic acid–Schiff (PAS) reaction and . with special methods, (the fibers appear black; thus, they are said to be argyrophilic ). * Reticular fibers are named for their arrangement in a meshlike pattern or network. - In loose connective tissue, networks of reticular fibers are found at the boundary of connective tissue and epithelium, as well as surrounding adipocytes, small blood vessels, nerves, and muscle cells. - They are also found in embryonic tissues. . The prevalence of reticular fibers is an indicator of tissue maturity. - They are prominent in the initial stages of wound healing and scar tissue formation. - Reticular fibers also function as a supporting stroma in hemopoietic and lymphatic tissues . . In these tissues, a special cell type, the , produces the collagen of the reticular fiber. - It surrounds the fiber with its cytoplasm, thus isolating the fiber from other tissue components. . In most other locations, reticular fibers are produced by fibroblasts. . Important exceptions to this general rule include - the endoneurium of peripheral nerves, where Schwann cells secrete reticular fibers, and - tunica media of blood vessels, and muscularis of the alimentary canal, where smooth muscle cells secrete reticular fibers. Reticular Fibers in lymphatic nod reticular stain, 75x Spleen reticular fibers. Reticular Fibers in Liver, reticular stain, 75x Reticular Fibers Kidney

Thyroid Gland

Amnion Reticular fibers. Elastic fibers * Elastic fibers allow tissues to respond to stretch and distension. - Description . Elastic fibers are typically thinner than collagen fibers (0.1-10 µm thick fiber) , . are arranged in a branching pattern to form a three dimensional network, and . High elasticity (rubber – like consistency) - The fibers are interwoven with collagen fibers to limit the distensibility of the tissue and prevent tearing from excessive stretching. - Special stain: . Resorcin fuchsin, Aldehyde fuchsin, Van Gieson (mordant hematoxylin), and Orcein. - Distribution: . Elastic connective tissue, Ligamentum flava, Vocal ligament, Stylohyoid ligament, Elastic membrane and lamellae of artery, and Elastic cartilage Elastic fibers Structure * Elastin, (72 kilodaltons) is a protein central (pars amorpha), rich in - Glycine (half), proline (10%), hydroxyproline, lacks hydroxylysine. . The random distribution of glycines makes the elastin molecule - hydrophobic and - allows for random coiling of its fibers. - Desmosine, isodesmosine; two large amino acids unique to elastin, which are responsible for the covalent bonding of elastin molecules to one another. * Microfibrils, peripheral ( Pars filamentosa), measuring 11to 13 nm in diameter, consist of -1 - Fibrillin (350 kilodaltons) is a : . No isodesmosine, desmosine, hydroxyproline, half cystine residue, 5% neutral sugar - fibrillin-1 microfibrils are used as substrates for the assembly of elastic fibers during elastogenesis. - The microfibrils are formed first; elastin material is then deposited on the surface of the microfibrils. - Elastin-associated fibrillin microfibrils play a major role in organizing elastin into fibers. Elastic fibers

* evolution - Oxytalan fiber . Microfibril . No elastin - fiber . Elastin deposition . Microfibril - Elastic fiber . Elastin, (central) . Microfibril, (peripheral) The diagram shows a small piece of an elastic fiber, in two conformations. Elastin polypeptides, the major components of elastic fibers, have multiple random-coil domains that straighten or stretch under force, and then relax. Most of the cross-links between elastin subunits consist of the covalent, cyclic structure desmosine, each of which involves four converted lysines in two elastin molecules. This unusual type of protein cross-link holds the aggregate together with little steric hindrance to elastin movements. These properties give the entire network its elastic quality. Elastic fibers Central elastin and peripheral microfibrils Elastic fibers Stages in the formation of elastic fibers can be seen by TEM. (a) Initially, a developing fiber consists of many 10-nm- diameter fibrillin microfibrils composed of molecular subunits secreted by fibroblasts and smooth muscle cells. (b) Elastin is deposited on the scaffold of microfibrils, forming growing, amorphous composite structures. The elastin molecules are also secreted by the fibroblasts and quickly become cross-linked into larger assemblies. (c) Elastin accumulates and ultimately occupies most of the electron-dense center of the single elastic fiber shown here. Fibrillin microfibrils typically remain at the fiber surface. Collagen fibrils, seen in cross section, are also present surrounding the elastic fiber. All X50,000. B and C, Electron micrographs of a smaller bundle of elastin fibers (stained black) that illustrate the number and relationship of the fibrillin fibers to the elastin fibers. Part B is a longitudinal section, and part C is a crosssection of a similar bundle. (From Pollard TD, Earnshaw WC. Cell Biology. Philadelphia, WB Saunders, 2004, p 481.) Scanning electron micrograph of an elastic fiber.

elastin (E), collagen fibrils (C), small fibrillin microfibrils (arrows)

Collagen and elastic fibers, mesentery, rat, Weigert’s ×160. Elastic fibers, dermis, monkey, Welgert’s ×160.

Elastic lamellae, elastic artery, monkey, Weigert’s ×80. * Marfan Syndrome - A disease known as Marfan syndrome is caused by a mutation in the fibrillin-1 gene. - There are several different , and mutations. - Marfan syndrome is particularly interesting in that the phenotype demonstrates variable expressivity. . Marfan syndrome is an autosomal dominant disease. . It is commonly associated with bone overgrowth. . About 10% of affected individuals develop emphysema. . Many individuals with Marfan syndrome have aneurysms of the aorta as well as heart valve defects. . It has long been thought that the defect in fibrillin-1 caused structural abnormalities in the structure and hence the function of elastin fibers, which have a “coating” of fibrillins. . Recent evidence strongly suggests the underlying cause of the connective tissue defect is that β) - normal fibrillin-1 binds the transforming growth factor–β (TGF family of cytokines, down-regulating their activities. . Mutant fibrillin-1 does not bind TGF-β, which leads to its overactivity, particularl during development. . This results in gross abnormalities in the structure of elastin- and fibrillin rich connective tissues. Defects in Fibrillin-1 Marfan syndrome Ground substance - Ground substance is the part of the extracellular matrix that occupies the spaces between the cells and fibers; it is; . a viscous, . clear substance with a slippery feel and . high water content. - It consists of : . glycosaminoglycans (GAGs), . proteoglycans, and . multiadhesive glycoprotein. - The size and structure of the three groups of molecules vary enormously. * GAGs are responsible for the physical properties of ground substance. - The GAGs are the most abundant heteropolysaccharide components of ground substance. - GAG represent long-chain unbranched polysaccharides composed of repeating disaccharide units. . The disaccharide units contain either of two modified sugars —N- acetylgalactosamine (GalNAc) or N- acetylglucosamine (GlcNAc)—and a uronic acid such as glucuronate or iduronate. - GAGs (except hyaluronan) are synthesized by connective tissue cells as a covalent, posttranslational modification of proteins called proteoglycans (mucopolysaccharides). For example, . heparin is formed by enzymatic cleavage of ; dermatan sulfate is similarly modified from . - GAGs are highly negatively charged because of the sulfate and carboxyl groups. . The high density of the negative charge attracts water, forming a hydrated gel. - The gel-like composition permits rapid diffusion of water- soluble molecules. - The rigidity of the GAGs provides a structural framework for the cells. - On the basis of differences in specific sugar residues, the nature of their linkages, and the degree of their sulfation, a family of seven distinct GAGs is recognized. Structure Glycosaminoglycans

Proteoglycans are composed of GAGs covalently attached to core proteins. - The linkage of GAGs to the protein core involves a specific trisaccharide composed of two galactose residues and a xylulose residue. . The trisaccharide linker is coupled through an O- glycosidic bond to the protein core that is rich in serine and threonine residues, allowing multiple GAG attachments. - Proteoglycans are remarkable for their diversity: . The number of GAGs attached to the protein core varies from 1 (i.e., ) to more than 200 (i.e., aggrecan). . A core protein may have identical GAGs attached to it (as in the case of fibroglycan or versican) or different GAG molecules (as in the case of aggrecan or syndecan). . Transmembrane proteoglycans such as syndecan link cells to ECM molecules

A schematic representation of a proteoglycan. It consists of a protein core molecule bound with many different types of Glycosaminoglycans (GAGs).

For example, a proteoglycan from cartilage matrix can have 30 keratan sulfate and 100 chondroitin sulfate GAG chains. Proteoglycan structure. This schematic drawing shows, on the right, a proteoglycan monomer and its relationship to the hyaluronan molecule as represented in the ground substance of cartilage. The proteoglycan monomer is composed of a core protein to which GAGs are covalently bound. The proteoglycan monomer consists of different numbers of GAGs joined to the core protein. The end of the core protein of the proteoglycan monomer interacts with a link protein, which attaches the monomer into the hyaluronan forming the proteoglycan aggregate. On the left, hyaluronan molecules forming linear aggregates, each with many proteoglycan monomers, are interwoven with a network of collagen fibrils. Common proteoglycan monomers of the connective tissue matrix.

Common proteoglycan monomers of the connective tissue matrix. Note the diversity of proteoglycan molecules; the number of GAGs attached to the protein core varies from one in decorin to more than 200 in aggrecan. Note also that versican has identical GAG molecules (chondroitin sulfate) attached to the core Ch molecule whereas aggrecan has a mixture of chondroitin sulfate and keratan sulfate attached to the core protein. Syndecan is a transmembrane proteoglycan that attaches the to the extracellular matrix.

Ch or De Proteoglycans Multiadhesive glycoproteins play an important role in stabilizing the ECM and linking it to cell surfaces.

- They are multidomain and multifunctional molecules that play an important role in stabilizing the ECM and linking cell surface. - They possess binding sites for a variety of ECM proteins such as collagens, proteoglycans, and GAGs; - They also interact with cell-surface receptors such as integrin and collagen XVII - Multiadhesive glycoproteins regulate and modulate functions of the ECM related to cell movement and cell migration as well as stimulate cell proliferation and differentiation. - Among the best characterized multiadhesive glycoproteins are the following: • Fibronectin • Laminin • • Osteopontin Common multiadhesive glycoproteins

Common multiadhesive glycoproteins. These proteins reside in the extracellular matrix and are important in stabilizing the matrix and linking it to the cell surface. They are multifunctional molecules of different shapes and possess multiple binding sites for a variety of extracellular matrix proteins such as collagens, proteoglycans, and GAGs. Note that multiadhesive proteins interact with basal membrane receptors such as integrin and laminin receptors. 20 different fibronectin molecules Multiadhesive Glycoproteins Connective tissue cells Connective tissue cells can be resident or wandering - The cells that make up the resident cell population are relatively stable; - They typically exhibit little movement and can be regarded as permanent residents of the tissue. These include: • fibroblasts and a closely related cell type, the , • Pigment cells • macrophages, • adipocytes, • mast cells, and • adult stem cells. - The population or transient cell population consists primarily of cells that have migrated into the tissue from the blood in response to specific stimuli. These include: • lymphocytes, • plasma cells, • neutrophils, • eosinophils, • basophils, and • monocytes. LCT – M = ; P = plasma cells; F = fibroblasts; Eo = eosinophils, N = neutrophils LM of fibroblasts (arrows) The fibroblast is the principal cell of connective tissue. Active (left) and quiescent (right) fibroblasts. External morphologic characteristics and ultrastructure of each cell are shown. Fibroblasts that are actively engaged in synthesis are richer in mitochondria, lipid droplets, Golgi complex, and rough endoplasmic reticulum than are quiescent fibroblasts (fibrocytes). SEM image of a long fibroblast cell projection in culture Fibroblasts in connective tissue. a. Photomicrograph of a connective tissue specimen in a routine H&E–stained, paraffin embedded preparation shows nuclei of fibroblasts (F ). 600. b. During the repair process of a wound, the activated fibroblasts (F) exhibit more basophilic cytoplasm, which is readily observed with the light microscope. 500. Fibroblasts Fibroblasts in connective tissue ( scanning electron micrograph Myofibroblast The myofibroblast displays properties of both fibroblasts and smooth muscle cells. - The myofibroblast is an elongated, spindly connective tissue cell. - It is characterized by the presence of bundles of actin filament (α-smooth muscle actin) with associated actin motor proteins such as nonmuscle myosin . regulated by TGF-1. - actin fibers attachment to the plasma membrane . serves as a cell-to-ECM anchoring junction and is called fibronexus. - This arrangement is the basis of a mechanotransduction system in which force that is generated by the contraction of intracellular actin bundles is transmitted to the ECM. - contains and dense bodies similar to those observed in smooth muscle cells - displays typical characteristics of the fibroblast along with characteristics of smooth muscle cells. - it lacks a surrounding . - its processes may contact the processes of other myofibroblasts by gap junctions. Electron micrograph of fibroblasts. The processes of several Electron micrograph of a myofibroblast. The cell fibroblasts are shown. The cytoplasm contains conspicuous profiles of exhibits some features of a fibroblast, such as areas with a rER. The cisternae of the reticulum are distended, indicating active moderate amount of rER. Compare with Figure 6.20. Other areas, synthesis. The membranes of the Golgi apparatus (G) are seen in however, contain aggregates of thin fi laments and cytoplasmic proximity to the rER. Surrounding the cells are collagen fibrils (CF), densities (arrows), features that are characteristic of smooth almost all of which have been cut in cross-section and thus appear as muscle cells. The arrowheads indicate longitudinal profiles of small dots at this magnification. 11,000. collagen fibrils. 11,000. Fibroblasts and myofibroblasts in the culture. of pigment connective tissue Melanocyte (pigment cells) of the iris * Adipocytes (Fat Cells) The is a connective tissue cell specialized to store neutral fat and produce a variety of hormones, not capable of mitotic division as adult cell 2 Types: a) White Fat, most common, serves as storage tissue for lipid (energy reserve). . During development accumulates lipid droplets which coalesce to form a large central droplet (unilocular); . cell therefore takes on "signet-ring" appearance – cytoplasm ( ring), flattened nucleus on periphery.

b) Brown Fat, contains multiple lipid droplets (multilocular), central nucleus, high numbers of mitochondria; brown color from high concentration of cytochrome in mitochondria. . Occurs only in mammals; relatively scarce, found in anterior regions of body (e.g., between scapulas) in neonates, hibernators and cold-adapted mammals. . functions in thermogenesis ( heat production) Brown and White adipose tissue Brown and White adipose tissue Adipocytes (fat cells) Cells of the Mononuclear phagocytic system Name of Cell Location Monocyte and its precursors in bone marrow: Monoblast and promonocyte Blood and bone marrow Macrophage Connective tissue, spleen, lymph nodes, bone marrow, and Perisinusoidal macrophage (Kupffer cell) Liver Alveolar macrophage Lungs Fetal placental antigen–presenting cell (Hofbauer cell) Pleural and peritoneal macrophage Serous cavities Osteoclast (originate from hemopoietic progenitor cells) Bone (originate from hemopoietic progenitor cells) Central nervous system Langerhans cell Epidermis of skin, oral mucosa, foreskin, female genital epithelium Fibroblast-derived macrophage (originate from mesenchymal cells) Lamina propria of intestine, of uterus Lymph nodes, spleen Multinucleated giant cells (e.g., foreign body giant cells, Langhans giant cells; originate from fusion of several macrophages) Pathological granulomas: suture granuloma, tuberculosis Macrophages Macrophages are phagocytic cells derived from monocytes that contain an abundant number of lysosomes. - In Connective tissue macrophages, also known as tissue histiocytes, - They derived from blood cells called monocytes. - They have phagocytic activity. - May be attached to collagen fibers (fixed) or loose within matrix (free) . When stimulated, fixed macrophages detach and migrate to sites of bacterial invasion or tissue injury. - Lysosomes are abundant . The surface folds engulf the substances to be phagocytosed. - The secretory products released by the macrophage include a wide variety of substances related to the immune response, anaphylaxis, and inflammation. - nucleus stains darkly and is indented; cytoplasm often round, but can be variable in shape. - Macrophages are antigen-presenting cells and play an important role in immune response reactions. . have specifi c proteins on their surface known as major histocompatibility complex II (MHC II) molecules that allow them to interact with helper CD4 T lymphocytes. - Macrophages arrive after neutrophils to the site of tissue injury and undergo differentiation. * Two functional types of macrophage - Classically activated macrophages (M1 macrophages) promote inflammation, the destruction of ECM, and apoptosis. . M1 macrophages elicit chronic inflammation and tissue injury. - When macrophages encounter large foreign bodies, they may fuse to form a large cell with as many as 100 nuclei that engulfs the foreign body. . These multinucleated cells are called foreign body giant cells (Langhans cells). - Alternatively, activated macrophage (M2 macrophage) works toward resolution of inflammation and promotes rebuilding of ECM, cell proliferation, and angiogenesis. . They are anti-inflammatory. . work toward resolution of inflammation. . They promote wound repair due to their anti-inflammatory, proliferative, and angiogenic activities. . M2 macrophages are involved in pathogenesis of allergy and asthma. Photomicrograph and electron micrograph of a macrophage. a. This photomicrograph shows several macrophages (M) in the connective tissue from the area of wound healing. They can be distinguished from other cells by the presence of an indented or kidney shaped nucleus (similar to monocytes in the blood vessels). Note several mature neutrophils (N) with segmented nuclei located in the connective tissue that surround (BV) filled with red and white blood cells in the center of the image. 480. b. The most distinctive EM feature of the macrophage is its population of endocytotic vesicles, early and late endosomes, lysosomes, and phagolysosomes. The surface of the cell reveals a number of finger-like projections, some of which may be sections of surface folds. 10,000. Electron micrograph of macrophage Mast Cells Mast cells develop in bone marrow and differentiate in connective tissue. - Large, rather round cells with basophilic granules in cytoplasm and central pale nucleus; similar to basophil, but may arise from bone marrow. - Usually found associated with blood vessels. - The cell surface contains numerous microvilli and folds. - mast cell granules can be displayed with basic dyes. . It stains the granules intensely and metachromatically because they contain heparin (highly sulfated proteoglycan ). . The surface of mature mast cells expresses a large number of high-affinity Fc receptors (FcRI) to which immunoglobulin E (IgE) antibodies are attached. - Two types of human mast cells have been identified based on morphologic and biochemical properties. . Most mast cells in the connective tissue of the skin, intestinal submucosa, and breast and axillary lymph nodes contain cytoplasmic granules with a lattice- like internal structure. . These cells contain granule-associated tryptase and chymase and are referred to as MCTC mast cells or connective tissue mast cells. . In contrast, mast cells in the lungs and intestinal mucosa have granules with a scroll-like internal structure. - These cells produce only tryptase and are termed MCT mast cells or mucosal mast cells. - Nearly equivalent concentrations of each type are found in nasal mucosa. - Mast cells are especially numerous in the connective tissues of skin and mucous membranes but are not present in the brain and spinal cord. . Although the meninges (sheets of connective tissue that surround the brain and spinal cord) contain mast cells, the connective tissue around the small blood vessels within the brain and spinal cord is devoid of mast cells. - The absence of mast cells protects the brain and spinal cord from the potentially disruptive effects of the edema of allergic reactions. - Most mast cell secretory products (mediators of inflammation) are stored in granules and are released at the time of mast cell activation. . basophilic granules that store chemical substances known as mediators of inflammation which divided into two categories: - preformed mediators ; . heparin = clearing of plasma lipids, blood anticoagulant; . histamine = vasodilatations, increased permeability of venules, mediates inflammation . Serine proteases (tryptase and chymase). - tryptase serves as a marker of mast cell activation. - Chymase plays an important role in generating angiotensin II in response to vascular tissue injury. . Eosinophilic Chemotactic factor-A and B - newly synthesized mediators . Leukotriene C (LTC4) * Similar to histamine, leukotrienes trigger prolonged constriction of smooth muscle in the pulmonary airways, causing bronchospasm. The broncho constrictive effects of leukotrienes develop more slowly and last much longer than the effects of histamine. Bronchospasm caused by leukotrienes can be prevented by leukotriene receptor antagonists (blockers) but not by antihistaminic agents. The leukotriene receptor antagonists are among the most prescribed drugs for the management of asthma; they are used for both treatment and prevention of acute asthma attacks. • Tumor necrosis factor (TNF-) is a major cytokine produced by mast cells. - It increases expression of adhesion molecules in endothelial cells and has antitumor effects. • Several interleukins (IL-4, -3 -5, -6, -8 and -16), growth factors (GM-CSF), and prostaglandin D2 (PGD2) are also released during mast cell activation.

- Mediators released during mast cell activation as a result of interactions with allergens are responsible for a variety of symptoms and signs that are characteristic of allergic reactions. Mast Cells Section of rat tongue. Several mast cells in the connective tissue surround muscle cells and blood vessels. Pararosaniline-toluidine blue (PT) stain. Medium magnification. TEM of a Mast Cell

Electron micrograph of a human mast cell. The granules (G) contain heparin and histamine. Note the characteristic scroll-like structures within the granules. M, mitochondrion; C, collagen fibrils; E, elastic fibril; N, nucleus. x14,700. Inset: Higher magnification view of a mast cell granule. x44,600. Mast-cell secretion. 1: IgE molecules are bound to the surface receptors. 2: After a second exposure to an antigen (eg, bee venom), IgE molecules bound to surface receptors are cross-linked by the antigen. This activates adenylate cyclase and results in the phosphorylation of proteins. 3: At the same time, Ca2+ enters the cell. 4: These events lead to intracellular fusion of specific granules and exocytosis of their contents. 5: In addition, phospholipases act on membrane phospholipids to produce leukotrienes. . The process of extrusion does not damage the cell, which remains viable and synthesizes new granules. . ECF-A, eosinophil chemotactic factor of anaphylaxis. Comparison of features characteristic of Mast Cells and Basophils

Characteristic Features Mast Cells Basophils Origin Hemopoietic stem cell Hemopoietic stem cell Site of differentiation Connective tissue Bone marrow Cell divisions Yes (occasionally) No Cells in circulation No Yes Life span Weeks to months Days Size 20–30 µm 7–10 µm Shape of nucleus Round Segmented ( usually bilobar) Granules Many, large, metachromatic Few, small, basophilic High-affinity surface receptors for IgE antibodies (FcRI) Present Present Marker of cellular activity Tryptase Not yet established Adult Stem Cells and Pericytes Niches of adult stem cells are located in various tissues and organs. - Cells residing within niches in various tissues and organs (excluding bone marrow) are called tissue stem cells. - Bone marrow represents a unique reservoir of stem cells. - In addition to containing HSCs , bone marrow also contains at least two other populations of stem cells: . multipotent adult progenitor cells (MAPCs) that appear to have broad developmental capabilities and bone marrow stromal cells (BMSCs) that can generate chondrocytes, osteoblasts, adipocytes, muscle cells, and endothelial cells. . Niches of adult stem cells called mesenchymal stem cells are found in loose connective tissue of an adult. - give rise to differentiated cells that function in the repair and formation of new tissue such as in wound healing and in the development of new blood vessels (neovascularization). The vascular pericytes found around capillaries and venules are mesenchymal stem cells. - Pericytes, also called adventitial cells or perivascular cells, are found around capillaries and venules. - pericytes express a cohort of proteins similar to those of stem cells in the bone marrow. - Pericytes are surrounded by basal lamina material that is continuous with the basal lamina of the capillary endothelium. - pericytes surrounding the smallest venules have cytoplasmic characteristics almost identical with those of the endothelial cells of the same vessel. - Pericytes associated with larger venules have characteristics of smooth muscle cells of the tunica media of small veins. - the distal portion and proximal portion of the same pericyte exhibit characteristics of endothelial cells and smooth muscle cells, respectively. - the pericytes play The role of mesenchymal stem cells in which culture. Undifferentiated Mesenchymal Cells Pericyte Electron micrograph of a small blood vessel. At the right a that is in intimate relation to the endothelium

Electron micrograph of a small blood vessel. The nucleus at the upper left belongs to the endothelial cell that forms the wall of the vessel. At the right is another cell, a pericyte, which is in intimate relation to the endothelium. Note that the basal lamina (BL) covering the endothelial cell divides (arrow) to surround the pericyte. 11,000.

BL Wandering or transient cells (Defense cells of the LCT) Plasma cells, and WBC WBCs - Most common types in CT are lymphocytes, Eosinophils and Neutrophils. Basophils Basophils that develop and differentiate in bone marrow share many features with mast cells. - Basophils are granulocytes that circulate in the bloodstream and represent less than 1% of peripheral white blood cells ( leukocytes). - Basophils develop and mature in the bone marrow and are released to the circulation as mature cells. - They have high-affinity Fc receptors for IgE antibodies on their cell membrane. - They participate in allergic reactions and together with mast cells release histamine, heparin, heparan sulfate, ECF, NCF, and other mediators of inflammation. Section of an inflamed intestinal lamina propria. Inflammation was caused by nematode parasitism. Aggregated eosinophils and plasma cells function mainly in the connective tissue by modulating the inflammatory process. Giemsa stain. Low magnification. Lymphocytes Lymphocytes are principally involved in immune responses. - Connective tissue lymphocytes are the smallest of the wandering cells in the connective tissue. - They have a thin rim of cytoplasm surrounding a deeply staining, heterochromatic nucleus. - Normally, small numbers of lymphocytes are found in the connective tissue throughout the body. - The number increases dramatically, however, at sites of tissue inflammation. Lymphocytes are a heterogeneous population of at least three major functional cell types: T cells, B cells, and natural killer (NK) cells. - At the molecular level, lymphocytes are characterized by the expression of specifi c molecules on the plasma membrane known as cluster of differentiation (CD) proteins. - On the basis of these specifi c markers, lymphocytes can be classified into three functional cell types. • T lymphocytes are characterized by the presence of the CD2, CD3, CD5, and CD7 marker proteins and the T-cell receptors (TCRs). - These cells have a long life span and are effectors in cell-mediated immunity. • B lymphocytes are characterized by the presence of CD9, CD19, and CD20 proteins and attached immunoglobulins IgM and IgD. - These cells recognize antigen, have a variable life span, and are effectors in antibody mediated (humoral) immunity. • NK lymphocytes are non–T, non–B lymphocytes that express the CD16, CD56, and CD94 proteins not found on other lymphocytes. - they destroy virus-infected cells and some tumor cells by a cytotoxic mechanism. Lymphocytes Plasma Cells Plasma cells are antibody-producing cells derived from B lymphocytes. - Rare in most CT, more common in salivary glands, lymph nodes, and hematopoietic tissue. - Plasma cells are a prominent constituent of loose connective tissue where antigens tend to enter the body (e.g., the gastrointestinal and respiratory tracts). - plasma cell has only limited migratory ability and a somewhat short life span of 10 to 30 days. - The plasma cell is a relatively large, ovoid cell (20 m) with a considerable amount of cytoplasm. - The nucleus is spherical and typically off set or eccentrically positioned. . The cytoplasm displays strong basophilia because of an extensive rER - produce large amounts of only one type of protein a specific antibody Portion of a chronically inflamed intestinal villus. The plasma cells are characterized by their size and abundant basophilic cytoplasm (rough endoplasmic reticulum) and are involved in the synthesis of antibodies. A large Golgi complex (arrows) is where the terminal glycosylation of the antibodies (glycoproteins) occurs. Plasma cells produce antibodies of importance in immune reactions. PT stain. Medium magnification. Plasma Cells The plasma cell. a. This photomicrograph shows the typical features of a plasma cell as seen in a routine H&E preparation. Note clumps of peripheral heterochromatin alternating with clear areas of euchromatin in the nucleus. Also note the negative Golgi (arrows) and basophilic cytoplasm. 5,000. b. Electron micrograph shows that an extensive rER occupies most of the cytoplasm of the plasma cell. The Golgi apparatus (G) is also relatively large, a further refl ection of the cell’s secretory activity. 15,000. Plasma Cells

Ultrastructure of a plasma cell. The cell contains a well-developed rough endoplasmic reticulum with dilated cisternae containing immunoglobulins (antibodies). In plasma cells, he secreted proteins do not aggregate into secretory granules. Nu, nucleolus. Eosinophils, monocytes, and neutrophils are also observed in connective tissue. - Rapidly migrate from the blood to enter the connective tissue, particularly neutrophils and monocytes in case of immune responses and tissue injury. - Their presence generally indicates an acute inflammatory reaction. - neutrophils migrate into the connective tissue in substantial numbers, followed by large numbers of monocytes. . monocytes then differentiate into macrophages.

- The eosinophil, which functions in allergic reactions and parasitic infections. - Eosinophils may be observed in normal connective tissue, particularly the lamina propria of the intestine, as a result of chronic immunologic responses that occur in these tissues. The end