Progression to Chronic/Granulomatous Inflammation

Progression to chronic/granulomatous inflammation occurs when the acute inflammatory response fails. Failure is characterized by the following:

Examples of agents and substances that often result in chronic inflammatory responses include systemic mycoses, such as Blastomyces dermatitidis and Histoplasma capsulatum; intracellular bacterial pathogens, such as Nocardia, Brucella, Mycobacterium, or Salmonella spp.; protozoa, such as Leishmania or Trypanosoma spp.; parasites, such as Toxocara larva or Habronema; autoantigens, such as those occurring in sperm granulomas or in autoimmune diseases like lupus erythematosus; and foreign bodies (plant awns, sticks, metals, asbestos, or suture material). Such agents continually induce the release of inflammatory mediators from indigenous parenchymal cells and leukocytes, leading to macrophage infiltration and activation; T-lymphocyte, NK cell, and perhaps mast cell or eosinophil infiltration; and fibroblast and endothelial cell proliferation. Some of the inflammatory cytokines, such as TGF-β, may interfere with regeneration of epithelial and parenchymal cells (see section on Wound Healing and Angiogenesis).

Healing by Fibrosis

Healing by fibrosis occurs after tissue injury in which there is necrosis of the tissue framework provided by stromal elements (connective tissue) and of the epithelial cells required to regenerate and successfully reconstitute the parenchymatous elements of the tissue. After necrosis, dead tissue and the acute inflammatory exudate are removed by macrophages (phagocytosis by cells of the monocyte-macrophage system), and the space is filled with fibrovascular tissue (granulation tissue) commonly seen in the healing process. Granulation tissue is eventually replaced by immature fibrous connective tissue that is poorly collagenized and then by mature connective tissue that is well collagenized, healing the wound and forming a scar (cicatrix). Structural integrity may be reestablished, but functional integrity depends on the extent of the loss of parenchymal cells. For example, with severe skin burns or extensive lacerations, dermal scarring eventually replaces lost dermal structures and to a limited extent restores structural integrity; however, the skin’s functional integrity is extremely limited because of the loss of adnexal glands, hair follicles, and scar tissue that reduces the range of motion in joints of the limbs and digits. The degree and extent of fibroblast and myofibroblast proliferation in such wounds largely depends on mediators such as TGF-β and IL-13 (see the section on Wound Healing and Angiogenesis).

Abscess Formation

Abscess formation (Fig. 3-21) occurs when the acute inflammatory response fails to rapidly eliminate the inciting stimulus and the enzymes and inflammatory mediators from neutrophils in the exudate liquefy the affected tissue and neutrophils to form pus. Myeloperoxidase enzyme in neutrophils contributes to neutrophil necrosis and liquefaction. The presence of myeloperoxidase is an evolutionary phenomena and reptiles and birds lack this enzyme and are therefore unable to liquefy neutrophils into pus. Abscesses can have a septic or sterile origin. Septic abscesses most commonly originate from bacterial infection, whereas sterile abscesses arise from incompletely degraded foreign bodies or from the failure of injected medications to be completely absorbed. Pyogenic bacteria, such as Staphylococcus and Streptococcus spp., commonly cause septic abscesses. They enter tissue hematogenously or by direct extension from the skin following trauma. The pus within an abscess can range in consistency from serous to purulent to caseous and in color from white, to yellow, to green, depending on the inciting agent/substance. The color of the exudate often depends on the pigment produced by the inciting bacterium and the species; for example, yellow exudates are caused by abscesses formed by Staphylococcus, Streptococcus spp., and Corynebacterium ovis; green exudate is caused by abscesses formed by Pseudomonas aeruginosa; and red exudate is caused by abscesses formed by Serratia marcescens.

After an acute inflammatory response has been established, the development of an abscess in this site consists of a collection of neutrophils admixed with cell debris, macrophages, and fibroblasts with variable infiltrates of lymphocytes. Experimentally, such a site can form in 2 to 3 days, depending on the agent/substance. Fibroblasts in this site begin to produce collagen and ECM proteins that can form into a “thin” vascularized connective tissue area. At this point, antibiotics can penetrate this area and enter the exudate. If the septic abscess persists, the initial “thin” connective tissue area surrounding the exudates can mature into a fibrous capsule, which is thick and largely impermeable, in an attempt to “wall-off” the exudates from normal tissue. A capsular wall takes weeks to form. Abscesses with this response can present serious problems in systemic (hematogenous) or local (topical diffusion) antibiotic treatments. In large abscesses with abundant pus, the pus itself may dilute the antibiotic and further prevent the drug from reaching the optimal concentration required to kill the bacteria. It is for these reasons that larger abscesses are often lanced to drain the pus. Sterile abscesses do not require antibiotics or other drugs to kill an inciting agent/substance, but they do require breakdown of the capsule by lancing or some other means.

Granulomatous Inflammation and Granuloma Formation

Granulomatous inflammation is a distinct type of chronic inflammation in which cells of the monocyte-macrophage system are predominant and take the form of macrophages, epithelioid macrophages (activated macrophages), and MGC. In granulomatous inflammation, the cells are dispersed as sheets of cells distributed at random (diffuse or lepromatous) within parenchymal and connective tissue planes (Fig. 3-22, A), whereas in a granuloma (tuberculoid granuloma), they are arranged in distinct masses or nodules (Fig. 3-22, B).

Granulomatous inflammation occurs secondarily in response to endogenous or exogenous antigens or idiopathically as in granulomatous meningoencephalitis of dogs. Development and regulation of granulomatous inflammation requires multiple factors: (1) the inciting agent, usually with indigestible, poorly degradable, and persistent antigens (e.g., Mycobacteria spp.); (2) the host immune response (e.g., T_H and macrophage response); and (3) the interplay of various cytokines, chemokines, and other proinflammatory and antiinflammatory mediators produced by cells within the chronic inflammatory lesion.

Classification of granulomatous inflammation by pathologists has evolved over the years because of the increased understanding of disease pathogenesis and advances in molecular biology. For simplicity, this chapter discusses two morphologic forms of granulomatous inflammation: diffuse (lepromatous) granulomas, which are currently thought to be consistent with a T_H2-biased immunologic response, and nodular (tuberculoid) granulomas, which are currently thought to be consistent with a T_H1-biased immunologic response. Both of these terms are derived from granulomatous lesions in humans and are increasingly becoming defined and seen as distinct, both immunologically and molecularly.

Sarcoids of Horses

Sarcoids of human patients are granulomatous-like lesions. In contrast, sarcoids of horses are not a correlate to sarcoids in humans. Sarcoids that occur in the skin of horses are not granulomas as occur in humans but are locally aggressive skin tumors, and the most common dermatologic neoplasm reported in horses. They are composed of proliferating fibroblasts and do not contain the numerous macrophages, lymphocytes, and plasma cells seen in human sarcoids. Bovine papillomavirus (BPV) types 1 and 2 and the major transforming protein, E5, are associated with equine sarcoids but appear not to produce infectious virions. E5 may contribute to the persistence of the virus and development of the lesion by downregulating major histocompatibility complex (MHC) class I expression and thereby reducing immunosurveillance. The mode of transmission of BPV infection has not been determined.

Eosinophilic Granulomas

Certain types of chronic inflammation have dense infiltrates of eosinophils with macrophages, and varying numbers of lymphocytes and plasma cells (Web Fig. 3-14). Because of the presence of eosinophils, they are termed eosinophilic granulomas (Table 3-8). Some granulomas with numerous eosinophils develop in response to migrating parasites, such as Toxocara canis (larval migrans). In eosinophilic granuloma of cats, eosinophilic stomatitis of dogs, and equine eosinophilic dermatitis of horses, it is suspected these conditions form in response to antigen in a T_H2-directed manner; however, no specific antigen has been identified.

Grossly, eosinophilic granulomas in cats appear as papules, nodules, plaques (sometimes linear), and ulcers in the skin. They also occur as nodular or ulcerated lesions in the oral mucosae and footpads. Microscopically, the inflammatory response consists of eosinophils, macrophages, and areas of dense eosinophilia around collagen (Fig. 3-26). For many years, the densely eosinophilic, collagen-rich areas were considered regions of collagen degradation; however, the eosinophilic material is composed largely of major basic protein (MBP), a protein present in large amounts in the granules of eosinophils. Apparently eosinophils degranulate in these regions, releasing MBP, which accumulates over time. Some eosinophilic granulomas do not form distinct nodular granulomas and the cellular content of the lesions (e.g., the numbers of macrophages and eosinophils) can vary widely. But the lesions are chronic in nature and contain enough macrophages and other chronic inflammatory cells to be classified as granulomas by most pathologists.

Other Chronic Inflammatory/Granulomatous Conditions

With chronic inflammation, organs can become enlarged by the granulomatous inflammation which, once formed, persists. For example, intense macrophage infiltration in the liver and spleen of Foxhounds with visceral leishmaniasis result in hepatomegaly and splenomegaly that can be detected clinically and at postmortem (Web Fig. 3-15). In other conditions, chronic inflammation results in chronic weight loss and debilitation. Dogs with inflammatory bowel disease often lack granulomatous inflammation within the intestinal mucosa, but instead the mucosa is characterized by chronic inflammation composed of lymphocytes, plasma cells, and macrophages within the lamina propria of the intestinal mucosa (Web Fig. 3-16). This lesion alters intestinal function that can result in severe weight loss and vomiting. Several conditions of humans and animals are characterized by chronic inflammatory lesions, but the cause of these lesions is poorly understood. One such condition, polyarteritis nodosa, is seen in humans and rats and only rarely in other species. It is characterized by perivascular infiltrates of lymphocytes, plasma cells, and macrophages. Dogs can develop idiopathic canine polyarteritis, a condition in which arterial lesions not only involve coronary arteries but also medium-to-small arteries of other organs. As indicated, the cause of eosinophilic granulomas has not been determined but is thought to occur secondary to parasitic larval migration, microbial infections, or foreign material. Finally, a variety of other human diseases are characterized by varying degrees of perivascular granulomatous inflammation but to date have no or very rarely seen veterinary correlates. These diseases occur around blood vessels, may be secondary to immunologic mechanisms, and include large-vessel vasculitis (giant cell [temporal] arteritis, Takayasu arteritis), medium-sized vessel arteritis (Kawasaki disease, [and polyarteritis nodosa]), and small-vessel vasculitis (e.g., Wegener’s granulomatosis, Churg-Strauss syndrome).

• Chronic inflammation composed of lymphocytes and plasma cells admixed with macrophages occurs commonly in the body (Fig. 3-27). Sometimes, lymphocytes and macrophages predominate over plasma cells, and such lesions can be called lymphohistiocytic. Histiocytic is a term used for macrophage infiltration; however, some pathologists use the term macrophagic. This type of inflammatory response is characteristically seen in the early stages of the chronic inflammatory response and in response to specific microbes, such as viruses, and in mucosal surfaces in response to antigenic stimulation.

• Fibrosing chronic inflammation is a region of chronic inflammation that is predominantly composed of fibrous connective tissue. This can occur in chronic traumatic pericarditis (hardware disease) of cattle where there are areas of pericardial fibrosis covered by fibrin and in the chronic fibrosis that surround necrotic regions of lung and form along the pleura of cattle with contagious bovine pleuropneumonia and chronic Mannheimia haemolytica pneumonia.

• Chronic-active inflammation has the same cellular components as chronic inflammation but also contains neutrophils, fibrin, and plasma proteins that are constituents of the acute inflammatory response. Chronic-active inflammation occurs when the inciting stimulus has not been removed from the exudate in the chronic inflammatory response, and it continues to elicit an acute inflammatory response. One should be careful not to confuse the morphologic diagnosis of chronic-active inflammation with the hepatic disease entity of dogs termed chronic-active hepatitis.

• Granulomatous inflammation has a basic cellular exudate consisting predominantly of activated macrophages and in some cases also epithelioid macrophages, multinucleate giant cells, and lesser numbers of lymphocytes and plasma cells. Granulomatous inflammation can be arranged in a diffuse or haphazard manner as seen in the thickened intestinal mucosa (i.e., lamina propria) of cattle with Johne’s disease (see Fig. 3-25). While the presence of a few macrophages in a lesion is indicative of chronic inflammation, these are considered to be granulomatous inflammation when the macrophages aggregate and begin to replace portions of the normal stroma.

• Pyogranulomatous inflammation has the same cellular exudate as granulomatous inflammation but also contains multifocal/random infiltrates of neutrophils, fibrin, and plasma proteins, which are constituents of the acute inflammatory response. Pyogranulomatous inflammation occurs when the inciting stimulus has not been removed from the exudate in the granulomatous inflammatory response, and it continues to elicit an acute inflammatory response. A nodular-like granulomatous area with neutrophils is termed a pyogranuloma (see later). Pyogranulomatous inflammation is often seen with infections caused by Blastomyces dermatitidis and is frequently multinodular. Pyogranulomatous is sometimes used loosely, with little discretion. For example, it can be used in lesions that are in transition from acute inflammation to chronic inflammation or during the “clean-up” phase of healing.

• Granulomas are a distinct type of granulomatous inflammatory response that occurs when macrophage infiltration is present in a well-defined area and thus form a distinct mass on gross observation. Granulomas can occur as noncaseating and caseating types, as described.

• A pyogranuloma is a nodular granuloma with a central area of neutrophils.

Lymphocytes

Lymphocytes play a key role in most chronic inflammatory lesions, especially in autoimmune diseases and in diseases with persistent antigen. As with macrophages, lymphocytes enter unresolved areas of acute inflammation within 24 to 48 hours, being attracted by chemokines, cytokines, and other stimuli. Histologically, they are often aggregated around blood vessels and surround granulomas or are distributed haphazardly within injured tissue (see Fig. 3-27). In viral encephalitides, lymphocytes are commonly distributed in a perivascular pattern, primarily in the gray matter. In other types of conditions, such as lymphoplasmacytic stomatitis and pododermatitis of cats, lymphocytes and plasma cells are the predominant cell types in the lesions.

γ/δ–T Lymphocytes

γ/δ–T lymphocytes are often the first type of T lymphocyte to arrive in the lesion of chronic inflammation and can contribute to the development of granulomas. This mechanism is supported by the fact that mice lacking γ/δ–T lymphocytes have defects in granuloma formation. However, the role of γ/δ–T lymphocytes in the establishment and persistence of granulomas has not been fully determined. Cattle have high numbers of circulating γ/δ–T lymphocytes, relative to other T lymphocytes, and are a useful model for assessing the role of γ/δ–T lymphocytes, in the formation of classic granulomas in Mycobacterium tuberculosis and Mycobacterium bovis infections and in the formation of diffuse granulomatous lesions in Mycobacterium avium-paratuberculosis infections.

α/β–T Lymphocytes (CD4/CD8)

α/β–T lymphocytes (CD4 and CD8 lymphocytes) enter areas of chronic inflammation and are key to the regulation of the type of adaptive immune response that ensues, albeit a T_H1, T_H2, or T_H0 response of chronic inflammation and/or granuloma formation. Under the influence of cytokines, these lymphocytes can further differentiate into (1) effector memory lymphocytes, which enter extralymphoid sites of inflammation; and (2) central memory lymphocytes, which settle into the blood and lymphoid organs. Memory lymphocytes contribute to the persistence of the chronic inflammatory response and granuloma formation.

Epithelium

Epithelial cells can contribute to chronic inflammatory responses in a variety of manners. For example, experimental models of granuloma formation have shown that Mycobacteria release an early secretory antigen-6 (ESAT-6), which induces release of MMP-9 from epithelia that is vital for recruitment of macrophage infiltration. Epithelia can also release type I interferons, cytokines, antimicrobial peptides, and chemokines and express adhesion molecules.

Mononuclear Cell Maturation and Trafficking in the Chronic Inflammatory Response

The macrophage is key to the development and persistence of chronic inflammation. Monocytes, derived from the bone marrow, form the monocyte/macrophage system by entering tissues and differentiating into macrophages (e.g., Kupffer cells, alveolar macrophages, and microglial cells) and are central to innate and adaptive immune systems. Recent work also shows that the spleen is a significant reservoir of monocytes, which can exit en masse on tissue injury. Monocytes are then recruited from the bloodstream to enter tissue and differentiate into macrophages that can also respond to tissue injury. Under noninflammatory conditions, the replenishment of tissue macrophages occurs through local proliferation and not via monocyte influx. However, with inflammatory stimuli, monocytes are recruited from the blood into tissue in response to inciting agents/substances.

In noninflamed tissue, monocytes expressing CX3CR1 and CCR5 chemokine receptors are attracted to tissues expressing their respective ligands (i.e., fractalkine (CX3CL) and MIP-1-a [CCL3]). In areas of inflammation, monocytes expressing CCR2 chemokine receptors are attracted by MCP-1 (CCL2). Attracted monocytes enter these areas in a manner similar to that described for the leukocyte adhesion cascade outlined for neutrophils. Slow rolling is mediated by E and P selectins and firm adherence by monocytes to endothelial cells is largely mediated by LFA-1 (CD11a/CD18), VLA-4 (α₄β₁-integrin), and also Mac-1 (CD11b/CD18) molecules that adhere to the respective endothelial cell ligands: ICAM-1/2, VCAM-1, and ICAM-1/2. Transmigration of monocytes between endothelial cells is mediated by leukocyte adhesion molecules expressed on the monocyte such as LFA-1, VLA-4, Mac-1, PECAM-1, and JAM A, JAM B, and JAM C. These molecules bind to adhesion molecules, such as PECAM-1, and the JAM molecules expressed by endothelial cells at the intracellular junction. As monocytes and other leukocytes pass between endothelial cells, they separate the tight junctions and vascular-endothelial (VE)-cadherins to allow the passage of the leukocyte.

T_H1, T_H2, T_H17, and T reg lymphocytes, along with inflammatory mediators released by somatic cells and macrophages during injury and/or infection, affect the differentiation of monocytes and noncommitted macrophages to macrophages with specific function (see Web Fig. 3-21). In conditions favorable to T_H1 influence, macrophages respond to IFN-γ released by T_H1, NK lymphocytes, and TNF from antigen-presenting cells to form classically-activated macrophages (Table 3-9). Under T_H2 conditions, macrophages respond to IL-4 released by T_H2 lymphocytes and granulocytes (e.g., mast cells, basophils) by forming wound-healing (tissue repair) type macrophages. Under T-reg secretion of IL-10 and other substances, such as immune complexes, prostaglandins, glucocorticoids, and apoptotic cells, macrophages differentiate into regulatory macrophages (antiinflammatory cells), which secrete IL-10 to suppress inflammation. Macrophages committed to the classical-activation, wound-healing/tissue repair, or regulatory pathways greatly influence the anatomic (histopathologic) structure and function of the chronic inflammatory response and granuloma formation (Fig. 3-29; Web Fig. 3-18).

Macrophages activated within lesions enter lymphatic vessels that drain into nearby lymph nodes via the afferent lymphatic vessels. In addition, some macrophages from lesions within the caudal body can enter the thoracic duct, which drains into the cranial vena cava or one of its branches. Macrophages within the head and neck can enter right or left tracheal lymphatic ducts. The right tracheal lymphatic duct empties into the cranial vena cava, whereas the left tracheal duct empties into the thoracic duct. Once in the blood, the macrophages are disseminated throughout the body.

The outcome of these different types of macrophage activation leads to the following specific responses (see Fig. 3-29):

In addition to T reg lymphocytes and regulatory macrophages, recent work has shown that sialylated IgG molecules can regulate macrophage function. This stems from the observation that certain types of chronic inflammation, particularly autoimmune conditions of humans, have responded favorably to polyclonal IgG given intravenously. The mechanism by which this therapy works is poorly understood. Recently, evidence suggests that the polyclonal IgG activity may be due to a subset of IgG molecules that are sialylated on the Fc chain of the IgG. The sialylated IgGs are thought to interact with sialic acid–specific receptors on regulatory macrophages and upregulate expression of Fc receptor IIB, which is inhibitory, on effector macrophages.

Formation of Epithelioid Macrophages and Multinucleate Giant Cells

Activated macrophages within tissues are relatively large cells histologically (20 to 25 mm in diameter) with abundant, often clear cytoplasm and a single, oval-to-polygonal, often slightly eccentric, reniform nucleus (Fig. 3-30). With time, activated macrophages can sometimes further differentiate into epithelioid macrophages and MGC (Fig. 3-31).

Both epithelioid macrophages and multinucleate giant cells often form in response to foreign bodies or persistent intracellular pathogens. The molecular mechanisms by which epithelioid macrophages and multinucleate giant cells form are poorly understood. It is a fascinating biologic phenomenon that requires membrane fusion and integration of the cytoplasm and nuclei of multiple cells. Studies in human patients with sarcoidosis, a special type of granulomatous inflammation in humans (see section on Sarcoidosis) have elucidated some of the important factors and conditions that contribute to MGC formation. Epithelioid macrophages are larger than activated macrophages. They have abundant cytoplasm, and the cell membrane occasionally assumes a polygonal to elongated shape, forming sheets, and thus can resemble to a limited degree squamous epithelium. These cells have diminished phagocytic capacity, but they contain large amounts of rough endoplasmic reticulum (RER), Golgi, vesicles, and vacuoles. These latter structures suggest that the main function of epithelioid macrophages involves extracellular secretion; however, the physiologic activity of epithelioid macrophages is poorly understood and requires additional investigation.

MGC are seen frequently in granulomatous inflammation. MGC are syncytial cells formed by the fusion of two or more activated macrophages into one large cell with two or more nuclei (see Fig. 3-31). These nuclei can be distributed in the cell in a haphazard manner or aggregated in the center of the cytoplasm. This form is called a foreign body type of MGC. The nuclei can also be arranged in a horseshoe-like semicircle at the periphery of the cell. This form is called a Langhans’-type cell. The Langhans’-type giant cell should not be confused with Langerhans’ cells, which are dendritic cells (see next discussion) of the skin. Although epithelioid macrophages, foreign body MGC, and Langhans’ MGC have distinct cellular morphology, their physiologic activity is poorly understood.

The process of MGC formation (macrophage fusion) is not fully understood. It requires that macrophages be present within a chronic inflammatory milieu and thus likely bathed in cytokines, such as IFN-γ, IL-3, IL-4, IL-13, and GM-CSF; pathogen factors, such as muramyl dipeptide, a peptidoglycan portion of bacterial cells walls; and other inflammatory mediators. In this close proximity, the membranes of adjacent macrophages express fusionogenic molecules such as DC-STAMP (a seven transmembrane receptor), β₁ and β₂ integrins, CD44 (hyaluronic acid receptor), CD47 (integrin-associated protein), macrophage fusion receptor (MFR), fusion regulatory protein (FRP-1; CD98), and P2X7 (a ligand-gated ion channel activated by ATP that forms a pore). The fusion process has similarities to osteoclast formation.

B Lymphocytes

B lymphocytes contribute to chronic inflammation in at least two major ways. B lymphocytes can (1) take up and present antigen and (2) differentiate into immunoglobulin-producing cells (plasma cells or immunocytes), which secrete immunoglobulins that bind to and opsonize antigens facilitating phagocytosis. B lymphocytes are present within chronic inflammatory lesions and granulomas. They also populate the medullary sinus of lymph nodes in which they produce immunoglobulin locally or leave the medullary sinus through efferent lymphatic flow.

Plasma Cells

Under appropriate stimuli, such as intense antigenic stimulation and B lymphocyte presentation of antigens, B lymphocytes differentiate into plasma cells, which can secrete immunoglobulins that bind to and opsonize antigens and facilitate phagocytosis. Plasma cells form within lymph nodes, mucosal surfaces, and wound sites. The bone marrow also contains a resident population of plasma cells, which can increase in certain disease conditions. Clusters of these cells must be differentiated from neoplastic accumulations as can occur with multiple myelomas. Bone marrow plasma cells can easily migrate into venular walls in the bone marrow to enter the vasculature. Similarly, plasma cells within the medullary sinus of lymph nodes can enter efferent lymphatic vessels and eventually drain into the blood; however, peripheral blood often contains few plasma cells. In the chronic inflammatory exudate, plasma cells are usually found mixed with lymphocytes and macrophages, although in lesser numbers. Plasma cells predominate in certain chronic inflammatory conditions such as inflammatory bowel disease of dogs and cats, lymphoplasmacytic stomatitis and pododermatitis of cats, chronic dermatitis of any domestic animal species, and interstitial nephritis of dogs and cats.

Eosinophils

Different types of chronic inflammatory conditions and granulomas contain a low to high number of eosinophils. Eosinophils are recruited into and stimulated to proliferate within chronic inflammatory exudates by several mediators, most notably IL-5 and eotaxin. In some chronic inflammatory conditions that contain eosinophils, such as asthma in humans, there is a T_H2 shift, resulting in increased concentrations of chemokines, such as eotaxin, in the tissue that contribute to the recruitment of additional eosinophils and exacerbates the T_H2 response. The same result is likely true for other as yet poorly characterized conditions such as the eosinophilic complex of cats, eosinophilic infiltrates in the base of the tongue of Siberian huskies and other dogs, eosinophilic enteritis in boxer dogs, and eosinophilic inflammatory lesions in the skin of horses. For these conditions, it may be that some type of persistent yet unidentified T_H2-inducing antigen is present locally.

Mast Cells

Mast cells have a central role in triggering acute inflammatory reactions. In chronic inflammation, mast cells tend to look similar to macrophages in H&E-stained tissue sections and therefore are often not considered to be a part of the chronic inflammatory lesion. However, special stains, such as a Giemsa stain, of chronic or granulomatous inflammation frequently reveal a surprisingly large number of mast cells identified by their characteristic metachromatic granules. For example, chronic lung lesions (e.g., fibrosis and alveolar epithelial hyperplasia) that develop following severe Mannheimia haemolytica pneumonia often contain increased numbers of mast cells and reduced levels of substance P fibers resulting in persistently altered immune responses.

The reason for the presence of mast cells in chronic inflammatory conditions likely relates to their production of proteolytic enzymes such as chymase and tryptase. Such enzymes likely help physiologically in remodeling and fine tuning components of the ECM. With persistent inflammation and fibrosis, there can be increased proliferation of mast cells. Increased mast cell numbers in such lesions occur by increased infiltration and also by increased proliferation of mast cells in situ. With severe inflammation, there can be loss of substance P fibers, and mast cells can respond to this loss by increasing their expression of c-kit, an important regulator of mast cell proliferation.

Natural Killer Cells

NK cells are present in chronic inflammatory lesions, but their role varies based on the characteristics of the inflammatory stimulus. NK cells can kill cells recognized as foreign without prior exposure to antigen and thereby lacking antigen specificity as required by T lymphocytes. NK cells are activated by type I interferons and IL-12 and can activate macrophages and dendritic cells, thus contributing to chronic inflammation. NK-T lymphocyte activation can be triggered by lipid antigens in the presence of CD1d and can contribute to autoimmune responses.

Endothelial Cells

Endothelial cells are essential for neovascularization of chronic inflammatory lesions. The process of angiogenesis (neovascularization) in chronic lesions is similar to that which occurs during wound healing (see section on Wound Healing and Angiogenesis) and is induced by hypoxia and release of endothelial cell growth factors, such as FGF, VEGF, and PDGF.

Endothelial cells are interconnected by tight junctions composed of occludins, claudin, and JAMs, as well as adherens junctions composed of VE-cadherins. As leukocytes migrate between endothelial cells, leukocyte adhesion molecules bind some of these intercellular molecules. For example, LFA-1 molecule (CD11-α/CD18) binds JAM A, VLA-4 molecule (α-4/β-1) binds VCAM-1 and JAM B, and Mac-1 (CD11b/CD18) binds JAM C to mediate leukocyte passage between endothelial cells. These molecules are especially important for the transmigration of monocytes and lymphocytes through endothelial cell junctions into sites of chronic inflammation, by providing a stable yet temporary site of attachment of leukocyte filopodia and lamellipodia.

The Effect of Inflammation on the Febrile Response and Other Activities

Cytokines such as IL-1, TNF, and IL-6, as well as high mobility group protein box-1 (HMGB-1) molecules are often produced during acute inflammatory responses. These inflammatory mediators, along with LPS (microbial factor) and other potentially toxic microbial or chemical molecules are important regulators of body temperature (fever), malaise, headache, confusion, anorexia, locomotion, and unconsciousness. Many of these actions occur through (1) the effects of these cytokines on sensory fibers in the vagus nerve, which extends to the brainstem, and (2) their effects on cerebral endothelial cells, perivascular microglia, and meningeal macrophages and the ability of the cells to induce COX-1, COX-2, and COX-3 activity for the formation and release of prostaglandins such as PGE₂. PGE₂ then activates various hypothalamic nuclei, including the ventromedial preoptic nucleus, paraventricular nucleus, solitary tract nucleus, ventrolateral medulla, and parabrachial nucleus, which regulate fever and the other clinical responses listed previously.

Unique Types of Inflammation

First and Second Intention Healing

First intention healing in skin occurs when the edges of a wound site are directly apposed and reattach and heal to each other rapidly (Fig. 3-35). Wounds lacking such close, intimate apposition are termed second intention healing. In the simplest type of wound, such as a cut or incision in the skin by a surgeon, there is initial hemorrhage from the damaged vasculature and retraction and constriction of blood vessels. In the wound area, there is deposition of fibrin, leakage of plasma proteins, clot formation, platelet aggregation, and neutrophil infiltration. The nature of repair depends on several factors, including the proximity of the cut edges to each other, the presence or absence of foreign bodies or infectious microbes, and the general health capacity of the animal to repair wounds. Under ideal conditions, such as in surgery, first intention healing is desired (see Fig. 3-35). First intention healing occurs in nonseptic wounds, whereas second intention healing occurs in septic wounds, with foreign bodies, or gaping wounds with nonopposed edges. If the wound healing process is disrupted or delayed, the process is shifted to second intention healing. First and second intention healing are also discussed in Chapter 17.

First Intention Healing

First intention healing occurs in 2 to 3 days in the skin, if the cut edges of a nonseptic wound are positioned in close proximity to each other by sutures or bandages. During this time, the hemorrhage, plasma proteins, and cell debris within the wound are phagocytosed and removed by macrophages, new blood vessels sprout and grow into the lesion, and the ECM is synthesized to fill the gap between the apposed tissue edges. With time (weeks), this stable interconnection in the dermis is replaced by collagen fibers that undergo continual maturation, thus providing the skin with near-normal tensile strength after wound healing. Concurrently, basal cells of the squamous epithelium will undergo hyperplasia and cover the defect in 3 to 5 days. This type of repair leaves little trace of the wound, except for perhaps mild fibrosis in the superficial dermis and loss of adnexa (e.g., hair follicles, sebaceous glands, and sweat glands) at the site of the wound. The tensile strength is nearly the same as that of adjacent tissue. First intention healing is the goal of the surgeon for repair of incision sites made during surgery.

Second Intention Healing

Second intention healing occurs when the cut edges of the skin, for example, are not brought into appropriate apposition for healing (see Fig. 3-35). In such wounds, connective tissue is haphazardly synthesized and arranged and there is little or no organization in the healing process; however, fibrous connective tissue fills the defect in the superficial and deep dermis. This disorganization can also delay or prevent the migration of epithelial cells that attempt to cover the surface of the wound and disrupt the orderly deposition of ECM in the wound. In addition, new fibrous connective tissue lacks adnexa (hair follicles, sebaceous and sweat glands). In some cases, fibrous connective tissue can form into granulation tissue (see later section) in which histologically, proliferating fibroblasts are arranged perpendicular to new capillaries, and the long axes of the new capillaries are arranged perpendicular to the surface of the skin (Fig. 3-36). Tensile strength of granulation tissue is diminished and the lesions can tear or split. Thus with second intention healing, the site can remain ulcerated, lack hair, and in some cases, the fibrous connective tissue can undergo continuous proliferation and protrude from the skin surface as a hyperplastic scar.

Impaired Wound Healing

In addition to spontaneously occurring impairments of wound healing, such as foreign bodies, infection, and neoplasms, certain other conditions can prevent or impair wound healing, even first intention healing. For example, altered deposition of collagen and ECM proteins can occur with osteogenesis imperfecta because of impaired production of type I collagen. Similarly, impaired synthesis, cross-linking, hydroxylation, or posttranslational processing of collagen can delay wound healing in individuals with Ehlers-Danlos syndrome. Hyperglycosylation of proteins, which can occur with prolonged diabetes mellitus, can alter the vasculature, lead to diabetic ulcers, and inhibit wound healing.

Chemotherapeutic drugs can also prevent cellular proliferation and may reduce healing. Several new chemotherapeutic drugs specifically target endothelial cell proliferation, which may greatly influence the process of neovascularization so vital to efficient wound repair. In humans, guinea pigs, and other species that require dietary vitamin C, deficiencies in intake can lead to scurvy, a disease in which there is decreased collagen hydroxyproline synthesis and poor wound healing. Extreme starvation, malnutrition, and cachexia from cancer or severe weight loss from chemotherapy can impair the synthesis and deposition of ECM proteins as a result of negative energy balance and a lack of amino acid substrates, normally synthesized in the liver. Additionally, such individuals and severe burn victims often lack adequate levels of serum proteins such as albumin, which results in lowered osmotic plasma pressure, impaired fluid resorption from the wound site, and enhanced edema fluid accumulation.

Expression of Genes Responsible for Wound Repair

Wound repair requires activation of genes of the viable cells such as macrophages, fibroblasts, and endothelial cells adjacent to the sites of tissue injury. As indicated, macrophages internalize through phagocytosis cell debris to “clean-up” an area and degrade the ECM. In concert with fibroblasts, macrophages release growth factors that enhance the proliferation of (1) endothelial cells for neovascularization, (2) fibroblasts for deposition of a new ECM, (3) myofibroblasts for wound contraction, and (4) parenchymal cells for return to normal structure and function of the affected tissue.

Gene expression by cells in a wound is regulated to a large degree by oxygen levels (Fig. 3-37; Web Fig. 3-22). In the milieu of a wound, there is generally a reduced oxygen tension caused by vascular damage. Normal tissues have oxygen levels >90% oxygen saturation, and there is increased activity of non-heme iron-containing 2-oxglutarate (2-OG)-dependent oxygenases that sense oxygen levels and use dioxygen as a cosubstrate. These include prolyl hydroxylase domain-containing protein-1 (PHD-1), PHD-2, PHD-3, and factor-inhibiting HIF (FIH). These enzymes place a hydroxyl group on proline and asparagine amino acids in HIF-1α protein. Hydroxylated HIF-1α is degraded by the ubiquitin pathway when oxygen levels are high. In hypoxic tissue, however, as occurs in wounds, within neoplastic masses, and areas of inflammation, there is reduced activity of PHDs and FIH and thereby less hydroxylation of HIF-1α. Nonhydroxylated HIF-1α aggregates with HIF-1β and induces transcription of hypoxia-responsive elements (HREs) in the genome.

The HREs include genes for growth factors, including VEGF, iron-binding proteins, regulators of apoptosis, erythropoiesis, angiogenesis, pH regulation, and glucose and energy metabolism. Early growth response gene-1 (EGR-1) is another transcription factor activated in wounds that leads to expression of growth factors and cytokines. Therefore both HIF-1α and EGR-1 activity in hypoxic conditions lead to increased cellular transcription that upregulates genes for energy (glucose transporters, hexokinase 1 and 2, lactate dehydrogenase, phosphofructokinase), endothelial and fibroblast proliferation (TGF-β, VEGF), and iron sequestration (ceruloplasmin, transferring receptor). These genes promote cell survival in hypoxic conditions, enhance cell proliferation, especially of cells vital to repair (endothelial cells, fibroblasts), and delay or alter differentiation of other cells (epithelia or parenchymal cells) until endothelial and fibroblast proliferation is well established.

Degradation of Cells and Tissue Components in Wounds

Wounds generally have a central core composed of (1) degenerate and/or necrotic cells, such as parenchymal cells, fibroblasts, and endothelial cells, as well as infiltrating leukocytes, such as neutrophils, platelets, lymphocytes, mast cells, and macrophages; (2) inflammatory products (cytokines, eicosanoids, chemokines, and their respective receptors); (3) serum proteins (albumin, acute phase proteins, complement); (4) clotting proteins (fibrin); and (5) ECM proteins and substances. Many of these cells and mediators need to be removed before optimal healing takes place. Phagocytic cells, such as neutrophils and macrophages, are very important in the clean-up process through phagocytosis of particulate matter and subsequent lysosomal degradation and the release of digestive enzymes into the tissue. In addition, macrophages have a major role in the uptake of apoptotic cells that form in response to TNF-α or other proapoptotic inflammatory stimuli. The ECM can be especially difficult to degrade. However, macrophages and fibroblasts are key to this process through the release of matrix metalloproteinases that degrade the ECM.

Resynthesis of the Extracellular Matrix with Wound Healing

Fibroblasts and the Mechanistic Basis of Fibrosis

Fibroblasts align along planes of tissue stress during development (Langer’s lines or tension lines). In quadrupeds, these lines are generally dorsoventral over the thorax and abdomen (axial body plane) and parallel to the long axis of the limbs (appendicular body plane). Surgical incisions along Langer’s lines extend between, rather than transect, bands of fibrous connective tissue and tend to pull the margins of surgical skin incisions together. Such incisions reduce the degree of postsurgical scar formation.

Fibroblasts of cats appear to be especially responsive to injury and inflammation. In fact, injury of fibroblasts has been associated with their neoplastic transformation in cats. For example, traumatic lens rupture can lead to intraocular inflammation and fibroblast proliferation, and in some cases, fibrosarcomas. In addition, fibroblast proliferation and fibrosarcomas are common in cats at vaccination sites.

Initially during the hemostasis and inflammation phases of wound repair, fibrin and serum proteins form a loose gel-like framework for the migration of fibroblasts and endothelial cells into the wound to form granulation tissue. Simultaneously, leukocytes and other cells, such as fibroblasts and endothelial cells, are stimulated by HIF-α and epidermal growth factor (EGF) to synthesize and release a variety of growth factors that result in fibroblast proliferation and migration. These factors include FGF-1 and FGF-2, PDGF, EGF, and TGF-β1, 2, and 3. FGF, PDGF, IL-13, and TGF-β induce fibroblasts to produce collagen, whereas FGF, VEGF, TGF-β, angiopoietin, and mast cell tryptase induce endothelial cells to proliferate and migrate and produce basement membrane for formation of new capillaries (Web Fig. 3-28).

With time, the newly formed, provisional connective tissue is remodeled into a more mature matrix. In the entire process, TGF-β has a central role in fibroblast activity and collagen deposition, because it is produced by platelets and macrophages and induces macrophage chemotaxis, fibroblast migration and proliferation, and synthesis of collagen and ECM proteins. TGF-β binds TGF-β receptor II (TGF-βRII), which dimerizes with TGF-βRI. The TGF receptor then phosphorylates R-SMAD and Co-SMAD to overcome inhibition of SMAD 7. This signaling process induces fibroblast activity, and regulation of the signaling may be useful in therapeutic strategies to control scarring and/or fibrosis (Web Fig. 3-29).

In addition to producing collagen, fibroblasts can migrate to a certain degree, and this process is mediated by adhesion molecules that bind to the ECM. This binding is a complicated event in which the adherence process is essential for migration of the cell and its anchoring to extracellular proteins. During wound repair, proliferating fibroblasts often align themselves parallel with lines of tension stress.

Morphology of Granulation Tissue and Fibrous Connective Tissue

Wound Contraction

Angiogenesis in Wound Repair

Angiogenesis is the formation of new blood vessels from preexisting vessels. It is a process essential for all living organisms with a cardiovascular system and involves a series of steps, as illustrated in Fig. 3-40, for the formation of new capillaries, including the following:

This process occurs because as wounds heal, new vessels are necessary to supply the injured site with oxygen, remove carbon dioxide and other waste products, drain excess fluid, and provide a vascular pathway for cells and stem cells into the wound. This same beneficial process has also been adapted by primary and metastatic neoplastic cells to grow and spread throughout tissues of the body.

Epithelialization in Wound Repair

Epithelialization (reepithelialization) is the process by which the skin and mucous membranes replace superficial epithelial cells damaged or lost in a wound. Epithelial cells at the edge of a wound proliferate almost immediately after injury to cover the denuded area. Under normal conditions, this process is rapid, and first intention healing occurs in 3 to 5 days to repair the wound. During wound repair, keratinocytes and mucosal epithelial cells must move laterally across the wound surface to fill the void. Before this lateral movement can occur, epithelial cells must disassemble their connections to the underlying basement membrane and their junctional complexes with neighboring cells. They must also express surface receptors that permit movement over the ECM of the wound surface.

Inflammation

Aderem, A, Ulevitch, RJ. Toll-like receptors in the induction of the innate immune response. Nature. 2000;406:782–787.

Anthony, RM, Nimmerjahn, F, Ashline, DJ, et al. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science. 2008;320:373–376.

Benko, S, Philpott, DJ, Girardin, SE. The microbial and danger signals that activate Nod-like receptors. Cytokine. 2008;43:368–373.

Cantor, JM, Ginsberg, MH, Rose, DM. Integrin-associated proteins as potential therapeutic targets. Immun Rev. 2008;223:236–251.

Charo, IF, Ransohoff, RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. 2006;354:610–621.

Etzioni, A. Leukocyte adhesion deficiencies: molecular basis, clinical findings, and therapeutic options. Adv Exp Med Biol. 2007;601:51–60.

Geddes, K, Magalhaes, JG, Girardin, SE. Unleashing the therapeutic potential of NOD-like receptors. Nat Rev Drug Disc. 2008;8:465–479.

Gibson-Corley, KN, Hostetter, JM, Hostetter, SH, et al. Disseminated Leishmania infantum infection in two sibling foxhounds due to possible vertical transmission. Can Vet J. 2008;49:1005–1008.

Gill, S, Wight, TN, Frevert, CW. Proteoglycans: key regulators of pulmonary inflammation and the innate immune response to lung infection. Anat Rec. 2010;293:968–981.

Helming, L, Gordon, G. The molecular basis of macrophage fusion. Immunobiology. 2008;212:785–793.

Kanneganti, T-D. Central roles of NLRs and inflammasomes in viral infection. Nat Rev Immunol. 2010;10:688–698.

Kawai, T, Akira, S. Toll-like receptor and RIG-1-like receptor signaling. Ann NY Acad Sci. 2008;1143:1–20.

Kenny, EM, Rozanski, EA, Rush, JE, et al. Association between outcome and organ system dysfunction in dogs with sepsis: 114 cases (2003-2007). J Am Vet Med Assoc. 2010;236:83–87.

Klune, JR, Dhupar, R, Cardinal, J, et al. HMGB1: Endogenous danger signaling. Mol Med. 2008;14:476–484.

Petersen, CA. Canine leishmaniasis in North America: emerging or newly recognized. Vet Clin North Am. 2009. (in press)

Soehnlein, O, Lindom, L. Phagocyte partnership during the onset and resolution of inflammation. Nat Rev Immunol. 2010;10:427–439.

Swirski, FK, Nahrendorf, M, Etzrodt, M, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 2009;325:612–616.

Thurmond, RL, Gelfand, WE, Dunford, PJ. The role of histamine H1 and H4 receptors in allergic inflammation: the search for new antihistamines. Nat Rev Drug Disc. 2008;7:41–53.

Yao, C, Sakata, D, Esaki, Y, et al. Prostaglandin E2-EP4 signaling promotes immune inflammation through TH1 cell differentiation and TH17 cell expansion. Nature Med. 2009;15:633–640.

Zabel, BA, Zuniga, L, Ohyama, T, et al. Chemoattractants, extracellular proteases, and the integrated host defense response. Exp Hematol. 2006;34:1021–1032.

Healing

Bonauer, A, Carmona, G, Iwasaki, M, et al. MicroRNA-92a controls angiogenesis and function recovery of ischemic tissues in mice. Science. 2009;324:1710–1714.

Braiman-Wiksman, L, Solomonik, I, Spira, R, et al. Novel insights into wound healing sequence of events. Toxicol Pathol. 2007;35:767–779.

Elmquist, JK, Scammell, TE, Saper, CB. Mechanisms of CNS response to systemic immune challenge: the febrile response. Trends Neurosci. 1997;20(12):565–570.

Faurschou, M, Borregaard, N. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 2003;5:1317–1327.

Foster, D, Parrish-Novak, J, Fox, B, et al. Cytokine-receptor pairing: accelerating discovery of cytokine function. Nat Rev Drug Discov. 2003;3:160–170.

Fraisl, P, Aragones, J, Carmeliet, P. Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory disease. Nature Reviews Drug Discovery. 2009;8:139–151.

Gangur, V, Birmingham, NP, Thanesvorakul, S. Chemokines in health and disease. Vet Immunol Immunopathol. 2002;86:127–136.

Giger, U, Boxer, LA, Simpson, PJ, et al. Deficiency of leukocyte surface glycoproteins Mo1, LFA-1, and Leu M5 in a dog with recurrent bacterial infections: an animal model. Blood. 1984;69:1622–1630.

Hong, HS, Lee, J, Lee, E, et al. A new role of substance P as an injury-inducible messenger for mobilization of CD29+ stromal-like cells. Nature Med. 2009;15:425–435.

Kehrli, ME, Jr., Ackermann, MR, Shuster, DE, et al. Animal model of human disease: bovine leukocyte adhesion deficiency: beta-2 integrin deficiency in young Holstein cattle. Am J Pathol. 1992;140:1489–1492.

Kilarski, WW, Samolov, B, Petersson, L, et al. Biomechanical regulation of blood vessel growth during tissue vascularization. Nature Med. 2009;15:657–662.

Li, L, Clevers, H. Coexistence of quiescent and active adult stem cells in mammals. Science. 2010;327:542–545.

Mayadas, T, Cullere, X. Neutrophil β₂ integrins: moderators of life or death decisions. Trends Immunol. 2005;26(7):388–395.

Mitsui, A, et al. Effects of fascial abrasion, fasciotomy, and fascial excision on cutaneous wound healing in cats. Am J Vet Res. 2009;70:532–538.

Palmer, MV, Waters, WR, Thacker, TC. Lesion development and immunohistochemical changes in granulomas from cattle experimentally infected with Mycobacterium bovis. Vet Pathol. 2007;44:863–874.

Ramirez-Romero, R, Brogden, KA, Gallup, JM, et al. Mast cell density and substance P-like immunoreactivity during initiation and progression of lung lesions in ovine Mannheimia haemolytica pneumonia. Microb Pathog. 2001;30:325–335.

Rottman, JB. Key role of chemokines and chemokine receptors in inflammation, immunity, neoplasia, and infectious disease. Vet Pathol. 1999;36:357–367.

Simionescu, M, Gafencu, A, Antohe, F. Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey. Microsc Res Tech. 2002;57:269–288.

Stappenbeck, TS, Miyoshi, H. The role of stromal stem cells in tissue regeneration and wound repair. Science. 2009;324:1666–1669.

Ulbrich, H, Eriksson, EE, Lindobom, L. Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharm Sci. 2003;24:640–647.

Volkman, HE, Pozos, TC, Zheng, J, et al. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science. 2010;327:446–469.

Wedemeyer, J, Tsai, M, Galli, SJ. Roles of mast cells and basophils in innate and acquired immunity. Current Opin Immunol. 2000;12:624–631.

Yang, D, Biragyn, A, Hoover, DM, et al. Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense. Ann Rev Immunol. 2004;22:181–215.