Granulomatous Diseases

Granulomas were first observed over 200 years ago during the autopsy of a tuberculous lung. The various current definitions of the granuloma are still based on histopathologic observations. The lesion is defined as a focal, chronic inflammatory tissue response composed mostly of macrophages and their derivatives the epithelioid cells, evoked by persistent, poorly degradable substances (1,3,9). In addition various granulomas may contain lymphocytes, sometimes plasma cells, giant cells, neutrophils, eosinophils, mast cells and fibroblasts. Based on the causative agent(s) granulomas have a diverse etiology. They are classified into 4 groups: 1) T cell-mediated immune granulomas formed to infectious agents. 2) Granulomas with unknown etiology but with a T lymphocyte-mediated profile. 3) Foreign body granulomas induced by inanimate substances. 4) Granulomas associated with malignant tumors (Table 1).

Table 1-4 shows a wide range of causative agents which induce chronic inflammations by their persistent irritation and/or immune stimulation.

Function of the Granuloma

The granuloma has a significant protective function. The replicating or inanimate agents cause chronic tissue irritations and evoke a programmed tissue inflammatory response that isolates and walls off the offender. This is especially useful in the case of replicating intracellular invaders (Mycobacteria, Listeria) that can disseminate throughout the body. The macrophages that converge at the site of bacterial invasion ingest the bacteria and intracellularly kill them. The compact structure of the granuloma and the efficient intracellular bactericidal activity successfully prevent the dissemination of the microorganisms. This is well illustrated by miliary tuberculosis (TB) where deficient granuloma formation allows the systemic spread of the bacilli. A negative facet of the granuloma is that the lesion may harbor within its burnt-out or calcified structure residual viable Mycobacteria, Histoplasma. These latent organisms may cause the flare-up of the disease decades later. In the helminthic infection schistosomiasis the liver granulomas that form around the parasite eggs shield the liver parenchyma cells against the secreted toxic substances.

Morphology and Cellular Composition of the Granulomas

Immune granulomas are composed of tightly packed mononuclear cells which can be delineated from neighboring tissues. Previously dermatologists emphasized the presence of palissading epithelioid cells as the hallmark of the hypersensitivity granulomas. These cells are large with ill-defined cytoplasmic borders containing eosinophilic cytoplasm and large pale ovoid nuclei. They derive from macrophages that had lost their phagocytic capacity. They may be present in T cell-mediated as well as in foreign body granulomas. An additional characteristic feature of the granulomas is the multinucleated giant cells generated by the fusion of activated macrophages. Giant cells may contain 20 or more nuclei arranged either peripherally (Langhans type) in immune or disordered (foreign body type) lesions. Whereas epithelioid cells are non-phagocytic but secretory, giant cells retain their phagocytic capacity and also produce and secrete cytokines. There is no functional difference between the Langhans or foreign body types of giant cells. In the prototypical T helper 1 (Th1) lymphocyte-mediated TB granulomas the macrophages, epithelioid cells are surrounded by T lymphocytes of the CD4+ and CD8+ subsets as well as γ/δ+ T cells. In the mature granulomas fibroblasts embedded within collagen fibers are found at the periphery (3). The prototypical T helper 2 (Th2) lymphocyte-mediated usually helminthic granulomas contain in addition to the macrophages, epithelioid cells, CD4+ and CD8+ T lymphocytes, also B lymphocytes at the periphery, mast cells and numerous eosinophils. The granulomas are also encased in fibrous capsules. Granulomas that form around foreign body irritants also contain macrophages, epithelioid and giant cells and pending the nature of the irritant some T lymphocytes, or in the case of the silica granuloma also plasma cells.

Mechanisms of Granuloma Formation

Granulomas are complex multicellular tissue reactions formed after a cascade of interactive systems that encompass adhesion molecule activity, capillary permeability, and chemokine, cytokine action. The exact nature and extent of the various components of this cascade are still being explored. Observations have been pieced together from animal models of granulomatous inflammations, biopsied material and bronchial lavage fluids of infected individuals. Historically, the prototype of the lung granuloma is the tubercle induced in infected individuals by M. tuberculosis bacilli. Inhalation of the bacilli triggers the non-specific innate immune response expressed chiefly by ingestion (phagocytosis) of the bacilli by alveolar macrophages and dendritic cells (28). Recognition of the alien bacterial surface is carried out by the ancient pattern recognition system that utilizes a number of receptors notably the Toll-like receptors (TLRs) that interact with mycobacterial lipoproteins(32). After the mannose receptors-mediated phagocytosis a cascade of intracellular signaling ensues and the key nuclear factor kappa B (NFĸB) is generated. This transfer factor is essential for the pro-inflammatory cytokines production. Cytokines are short peptides that serve as signaling agents between cells. The major cytokines include tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1), interleukin-12 (IL-12). Transmembrane TNFα inhibits the intracellular growth of the bacilli (52) and induces CCL5 (RANTES) chemokine (chemoattractant peptide) secretion in macrophages which attracts the Th1 subset of lymphocytes to the site of the invasion. Additional chemokines MCP-1, MIP-1α attract blood monocytes from the circulation. This results in the accumulation of maturing macrophages bound by hyaluronic acid (kernel of the granuloma). Such macrophages are capable to exert limited microbicidal effect on ingested bacilli. Neighboring innate defense γ δ receptored T cells and natural killer (NK) T cells also contribute to the inflammatory cytokine, chemokine cascade. The dendritic cells that had engulfed the bacilli upregulate their chemokine receptors and under the influence of CCL19, CCL20 and CCL21 chemokines secreted by the lymph node stroma and the high endothelial cells in the venules migrate to the regional lymph node. En route, the ingested bacilli are killed degraded and the bacillary peptide fragments are displayed on the membrane of the dendritic cells within the grooves of the major histocompatibility complex II (MHC II) molecules. Within the lymph node dendritic cell and T cell are in close contact by means of adhesion molecules, and the process of antigen presentation to the clonally selected T cell α/β receptors ensues. Continued secretion of IL-12 by the dendritic cell induces the differentiation of the T cell clones to the Th1 subset. Having undergone the process of sensitization the Th1 cell secretes IL-2 cytokine that promotes T cell survival and proliferation. The sensitized Th1 cells are attracted by a gradient of a number of chemokines such as CXCL8,-9,-10,11 to the site of the focally aggregated macrophages which contain the ingested bacilli. Focally secreted chemokines CCL2,-3,-4,-5 recruit fresh monocytes from the circulation and by the membrane-bound TNFα signal adhesion molecule expression on the converging cells is increased and the early granuloma now organizes into a characteristic structure(5,34). The CD4+ and CD8+ T cells arrange at the periphery. Some of the CD4+ cells are interspersed with macrophages which contain ingested bacilli (40). Thus, at the first step the invaders are sequestered by granuloma macrophages. Fibroblasts at the periphery of the compact lesion produce collagen bundles. The fibrotic capsule delineates the lesion and ensures that the bacteria are walled off from the surrounding tissue. The mature granuloma also shows neovascularization. The antigenically and IL-12 stimulated CD4+ Th1 type lymphocytes secrete IFNγ that activates the macrophages to acquire the microbicidal function. Such activated cells produce oxygen (hydrogen peroxide H2O2, hydroxyl radical HO•, superoxide anion O2-, singlet oxygen O2•) and nitrogen (NO nitrogen oxide) breakdown products (ROS and RNS reactive oxygen and nitrogen species respectively) that are strongly oxidative. Currently NO is considered to be the principal agent that kills the M. tuberculosis. Continued activation of the macrophages is also provided by TNFα secreted by both macrophages and Th1 cells. The CD8+ T cells also participate in protection by killing the bacilli-overloaded macrophages and releasing bacilli to be ingested by fresh activated macrophages (15, 49, 50, 51, 56).

The hallmark of the mature TB and other granulomas is the epithelioid cells that occupy the center of the lesions. These cells are transformed macrophages that had lost their phagocytic function but retained their secretory activity producing TNFα, IL-10 and the regulatory TGF-β cytokines. Activated macrophages occasionally fuse together to create multinucleated giant cells. Such fusion was shown experimentally to be induced by IL-1,-3,-4,-6 and GM-CSF cytokines all of which may be produced by the granulomas. Giant cells within the granuloma retain their phagocytic and microbicidal activity and secrete IL-1 α, TNFα and TGF-β cytokines contributing to the granuloma cellular dynamics (26).

The prototypic T helper 2 (Th2) lymphocyte-mediated granuloma is induced by Schistosoma mansoni worm eggs. These eggs are produced by female worms that live in the mesenteric venous plexus of the infected host. The eggs disseminate and lodge within the microcapillaries in the liver and intestines. The eggs are ~ 150 μm in length cannot be phagocytized. By secreted antigens they induce a specific immune T helper cell response that generates the granulomatous reaction around each egg. Granulomas that form around the eggs also serve a protective function, because the cellular layers shield the hepatic cells against the toxic effect of some egg components. It appears that glycoconjugates of the worm eggs interact with host dendritic cells and by triggering their Toll-Like Receptors (TLRs) can induce a biased Th2 response (19). This response generates the production of cytokines, chemokines which recruit and activate inflammatory cells different from those present in the TB type granuloma. The resultant egg granuloma is composed of macrophages, B cells, CD4+ CD8+ T cells, fibroblasts and many eosinophils. Information on the mechanism of granuloma formation derives from murine experiments. During lung granuloma formation around injected egg antigen-coated beads the innate response expressed IL-1β, TNFα, GM-CSF, IL-3, IL-6 cytokines and numerous chemokines both from the CCL (MCP-1, 2, 3, MIP-2, α, β) and CXCL 2,5,9,10,11 families needed for cellular recruitment. The evolving adaptive immune response showed an initial Th0 profile with IL-2, IFNγ, IL-4, IL-5 and IL-10 expression. By day 16 this response was polarized to the Th2 response with elevated IL-5, IL-13 expresssion (12). Studies with injected live eggs showed that after an early Th1 profile expressing IFN-γ, TNF-α, IL-4 and IL-2 cytokines a shift to the Th2 response occurs with IL-4, IL-5, IL-6, IL-13 expression (57). Just as histopathology may not differentiate between foreign body and T cell-mediated lesions, the molecular mechanisms that generate the two types of lesions may not basically differ from one another. In both responses recruitment from the circulation of monocytes or other inflammatory cells and their attraction to the site of irritation/infection is mediated by chemokines and cytokines. The magnitude and duration of the inflammatory response depends on the surface characteristics of the foreign body. Large non-phagocytizable particles such as surgical sutures, thorns, graphite, plastic beads are usually more inert and will evoke transient small granulomas. Ingested particles especially silica are powerful irritants within macrophages.They induce tissue-damaging reactive oxygen species (ROS) and reactive nitrogen species (RNS).The pathogenic potential of the particles depend on the varieties of the silica surface (47). Inhalation of quartz or other silica particles may lead to silicosis, a chronic granulomatous disease accompanied by pulmonary fibrosis and pulmonary hypertension (22). It may sometimes lead to the development of autoimmune responses due to the adjuvant action of silica particles on the immune system. Regardless of the surface properties of the foreign irritant tissue macrophages secrete several chemokines among them MCP-1, MIP-1α, MIP-1β, MIP-2 and RANTES that attract blood monocytes and lymphocytes to the site. Concurrently, IL-1β and TNFα cytokines are produced which amplify chemokine production, vascular permeability and macrophage aggregation. Some macrophages transform to epithelioid cells and fuse to create giant cells. The cellular response usually peaks during the early days of granuloma formation and subsides usually leaving behind a fibrous capsule. When large amounts of collagen fibers are deposited within the granuloma a dense fibrous tissue forms and cells die out.

Pathogenesis of the Granulomas

Two factors contribute to the pathogenesis of the granulomatous process. 1) When the inducer agent is a pathogen/invader or foreign body the intrinsic toxicity of the agents can damage the tissues. 2) The vigorous immune-inflammatory T cell-mediated response evoked by the pathogen recruits macrophages and other cells that during the activated stage and phagocytosis secrete tissue-damaging substances. Examples for the former are found in M. tuberculosis that has a waxy lipid cell wall constituent the cord factor (trehalose 6, 6’-dimycolate) which regulates in vitro the rope-like growth of the bacteria. This factor directly damages macrophages or lung cells and when injected into animals causes granulomatous inflammations (29). An additional component is muramyl dipeptide that is also granulomagenic. Both components trigger the innate immune response with cytokine (TNFα, IL-1α, IL-6, IL-10, IFNγ and chemokine CCL2 production.Mycobacterium leprae the causative organism of leprosy lives intracellularly in the skin, nasal mucosa and Schwann cells of the peripheral nerves. As a consequence there is gross thickening of the facial skin, hypopigmentation and loss of sensation to heat, cold and pain (2,6,30). The Gram- bacillus Brucella rapidly multiplies in the lymph node and causes probably by the Lipid A component of its endotoxin destruction of the lymphoreticular organs. When it colonizes the heart tissue it can cause fatal endocarditis. The Gram+ Listeria secretes the exotoxin Listeriolysin O• a β hemolysin that destroys red cells, neutrophils and monocytes. The fungus Histoplasma has tropism for the mucous membrane in the mouth where it causes lesions. All the quoted pathogens can sustain their intra-macrophage survival by subverting the killing machinery of the cells (arrest of the phagolysosomal fusion, disruption of signaling pathways) thereby assuring the chronicity of the infection. It should be pointed out that because of the intimate relationship between host and invading pathogen it is difficult to clearly isolate the pathogen or host-derived factors in the pathogenesis of the diseases.

As will be discussed in the next section the vigorous T cell-mediated response that develops after the antigenic stimulation is a major contributor to the pathogenesis of the disease. The CD4+ Th1 lymphocytes secrete IFNγ that activates macrophages. The acquisition of the microbicidal capacity induces the release of ROS and NOS breakdown products as well as the release of proteolytic enzymes which greatly contribute to innocent bystander cell destruction and collateral damage.

An example for the foreign body-induced pathogenesis is inhaled silica (mostly quartz) particles that are strongly irritating and cytotoxic to macrophages that engulf them.

Pathology of the Granuloma

In the immune granulomatous inflammation the strong sustained T lymphocyte-mediated inflammatory response is largely responsible for the ensuing pathology. This response recruits and activates inflammatory leukocytes that converge around the site of the invasion and phagocytize the invader pathogen.

The pathology of the mature granuloma is caused by several factors: 1) It is a space-occupying lesion that physically impinges on the neighboring tissue. 2) Leakage from activated macrophages of cytokines (TNFα), oxygen (H2O2, O2­, OH•, O2•) and nitrogen (NO) derivatives and proteolytic enzymes that damage the neighboring tissue. 3) Caseous necrosis. In some, especially the M. tuberculosis-induced granulomas the centrally located macrophages die by a variety of causes which comprise the toxic effect of M. tuberculosis products, loss of vascularization, lack of oxygen, the CD8+ T cell-mediated lysis of bacilli-laden macrophages and TNFα-induced apoptosis (programmed cell death) (15,49). The necrotic center is devoid of M. tuberculosis. Apparently the vigorous macrophage activity achieved its goal (56). Such necrosis is never observed in the sarcoid lesions. If the outer wall of the granuloma is well-vascularized the lesion involutes. In contrast low vascularization promotes cavity formation and severe tissue pathology (56). 4) In progressive disease the caseous necrosis of the M. tuberculosis granuloma proceeds due to proteolytic activity to liquefactive necrosis, the surrounding lung tissue lyzes and cavities form. Further erosion of the neighboring blood vessels and airways facilitates the spread of the bacilli. Cavitary tuberculosis is dangerous, it abrogates the protective function of the granuloma, the bacilli spread within the host and by cough and droplet infection disseminate to the outside environment (15). 5) As a consequence of intragranulomatous cytokine (TGF-β, TNFα, for Th1 lesions, IL-4, IL-13 for Th2 lesions) activity fibroblasts or myofibroblasts get activated and lay down collagen fibers which coalesce to a dense fibrotic tissue. With the elimination of the pathogen or the antigenic stimulus most granulomas burn out by fibrosis. When the healing process is normal minimal residual scarring is left behind. Locally secreted TGF-β also induces in macrophages or fibroblasts the production of TIMPs (tissue inhibitors of metalloproteases) that block the degradation of the deposited collagen fibers (7). The burnt-out foci of the M. tuberculosis tubercle then undergo calcification which can be observed in chest x rays. Unlike the TB granulomas, the sarcoid or schistosome worm egg-induced granulomas never calcify (Table 2). Overt fibrosis that occurs in the granulomatous lungs or livers then derange organ function. Excessive tissue fibrosis can cause pulmonary or portal hypertension and death in sarcoidosis, or schistosomiasis respectively. Suppuration at the center of the granulomas also occurs in deep fungal infections (blastomycosis,coccidioidomycosis) and lymphogranuloma venereum. Granuloma annulare a benign dermal inflammation shows necrobiotic degeneration of dermal collagen, surrounded by palissading inflammatory cells (35,55). In humans the schistosome egg induced immunopathology is manifested by postgranulomatous liver and intestinal fibrosis (14,41). Here the balance between the Th1-Th2 responses comes into play. Most infected individuals suffer from a milder intestinal form of the disease. However about 5-10 percent of the patients develop the hepatosplenic manifestations with severe hepatic (Symmers’) fibrosis, portal hypertension, ascites fluid formation, gastrointestinal bleedings and occasional death. In several studies the complexity of various contributory factors to immunopathology emerged. Hepatosplenic disease in Kenya was associated with high IFN-γ, TNF-α and low IL-5 cytokine production. Such profile is observed in the Th1 type TB granuloma. Thus results indicated that inability of a patient to develop a strong Th2 response leads to disease (43).Further research revealed that periportal fibrosis is age and gender dependent and induced by high TNF-α production (8). This conclusion remains controversial because a recent Brazilian study found that hepatic fibrosis at the early hepatosplenic stage is associated with high Th2 type cytokine chiefly IL-5, IL-10 and IL-13 production (14). This latter observation is in accordance with murine experiments that clearly showed impaired granuloma formation and liver fibrosis in mice defective in type 2 IL-4, IL-13 cytokine production. Thus in the clear cut murine system the Th2 response appears to dampen the early tissue-destructive IFN-γ mediated inflammation (54), yet it contributes to the pathology by IL-4, IL-13 cytokine-mediated liver fibrosis (60).

DIFFERENTIAL DIAGNOSIS

Granulomas are caused by an extremely broad range of disease processes. These can be conveniently divided into infectious and non-infectious causes. One of the difficulties in arriving at a diagnosis is that many etiologies have similar clinical syndromes. However, specific tests may help in distinguishing the two types of granulomas.

Infectious Causes

Although many infections are associated with granuloma formation, relatively few microorganisms cause the majority of cases. Mycobacteria and fungi are commonly associated with granulomatous infection, and in particular,tuberculosis is the most common cause of granulomas worldwide. However, all mycobacteria can be associated with granulomas. Most clinically important fungi are also associated with granuloma formation, including Aspergillus, Cryptococcus,Candida, and Histoplasma to name a few. Other important causes of granulomas are parasitic infections (schistosomiasis, leishmaniasis, dirofilariasis, etc.) and rarely, viral infections caused by cytomegalovirus, Epstein-Barr virus and measles(Table 1-4). Relatively few bacterial infections typically cause granulomas during infection, including brucellosis, Q-fever, cat-scratch disease (33) (Bartonella), melioidosis, Whipple’s disease (20), nocardiosis and actinomycosis. Tuberculosis can often be distinguished by its tendency to produce caseation necrosis within granulomas, though the other infectious etiologies are almost impossible to differentiate from each other based solely on histologic appearance (Table 3). One common thread among these disparate infectious syndromes is their general chronicity, many typically associated with weeks, even months of clinical symptoms before diagnosis is made. In addition, therapy is usually prolonged, for months to years before clinical resolution.

Non-Infectious Causes

Whereas infections can often be confirmed with bacterial cultures of affected tissues, non-infectious causes of granulomas may be difficult to diagnose, and often remain a diagnosis of exclusion.  Berylliosis is a T lymphocyte-mediated non-necrotizing granulomatous disorder that develops in beryllium metal-exposed workers(39). Sarcoidosis is one of the most common non-infectious granulomatous diseases, characterized by non-necrotizing epithelioid granulomas with giant cells in multiple organ systems, primarily the lungs(4,18,30). Over the years mycobacterial and propionibacterial organisms have been proposed as etiological agents of the disease.  A recent study detected M. tuberculosis catalase-peroxidase protein within sarcoidosis tissues providing further indication for the infectious etiology of the disease (42). Crohn’s disease is characterized by non-caseating ileal and colonic granulomas (Table 3).  Mucosal permeability to luminal pathogens among them M. paratuberculosis have been proposed as pathogenic factors(48,59). Another group of diseases are the vasculitis syndromes (46), which include Wegener’s granulomatosis, Churg-Strauss disease, and Takayasu’s arteritis to name a few. These usually cause necrotizing granulomas (18).Rheumatoid pulmonary nodules are often associated with granulomas.  In the liver, autoimmune diseases such as hepatitis and primary biliary cirrhosis are often characterized by granulomatous pathology (17).

Foreign bodies such as talc, silicone, surgical sutures may also elicit granulomatous reactions with less complex histological appearance.  Often the clinical presentation is organ specific such as renal granulomas caused by antibiotics, anti-inflammatory agents,diuretics(31).In the lung, aspiration of vegetable matter that remains under-graded can evoke granulomatous response.

Approach to Determining Etiology

As discussed above, the differential diagnosis of granuloma formation is very broad, and it can be difficult to arrive at a specific etiology. This is critical, as therapeutic pathways diverge from infectious to non-infectious causes, with almost all non-infectious causes requiring prolonged use of high dose steroids, whereas infections require specific long-term antimicrobial therapy for months and even years.

LABORATORY DIAGNOSIS

Laboratory diagnosis is an indispensable aid in the diagnosis of the granuloma etiology.  Presently, a wide range of laboratory methodologies is available for the correct diagnosis of the granulomatous condition.  This includes the histopathological examination of the granulomatous tissue, intragranulomatous localization and identification by staining, immunohistochemistry, immunofluoroscence of the putative pathogen and its in vitro cultivation.  Recent advances in molecular biology, proteomics (42) provided further sensitive and rapid tests for the correct diagnosis.  The immune etiology of the granulomatous diseases can now be confirmed by testing patient’s serum or T lymphocyte responses to the suspected granulomagenic organisms. The novel methodologies require expensive diagnostics, reagents, sophisticated instrumentation and expertise. In the following sections the methodologies will be described (Table 4).

Cultures

To establish the infectious etiology of the granuloma, cultures are prepared from homogenized biopsied tissues.Care must be exercised to rigorously exclude an accidental contaminant in the sample.  Growth of the pathogens(bacteria, fungi) on selective and/or differential solid medium provides information on the Gram staining properties: purple for Gram+,pink for Gram- of the pathogenic bacteria, as well as the morphology ,color of the colonies, biochemical activity and antibiotic resistance of the organisms.  Final identification of the pathogens taken from the isolated colonies can be made by specific antibodies.  Using enriched media: sheep blood agar, chocolate agar, brain heart infusion agar many organisms can be grown overnight.  However several classical granulomagenic organisms such as Mycobacteria, Brucella, Listeria or Histoplasma grow slowly and final diagnosis may be made only after 7-30 days.  This is a disadvantage when there is an urgency for a quick diagnosis. The actual choice of the biopsied tissue taken is also decisive for success. The highest percentage of M. tuberculosis positive cultures was obtained from samples with necrotic granuloma centers, whereas poorly formed granulomas yielded much lower positive cultures. Burnt-ot fibrotic lesions were usually culture negative.

Culture of Mycobacterium = Tuberculosis

For primary isolation of the bacteria special media are required. Many laboratories prefer the egg-based Lowenstein-Jensen solid medium on which bacteria may take 2-4 weeks to grow.  The American Trudeau Society medium and the Middlebrook 7 H10 solid or liquid media can also be used. The latter may yield a more rapid growth of the bacteria (24).  Many laboratories prefer the commercial automated BACTEC MGIT (Mycobacteria Growth Indicator Tube) 960 system in which bacterial growth in hundreds of inoculated tubes can be monitored. Growth of acid fast bacteria alone does not provide the final identification for M. tuberculosis.  This is accomplished by using molecular biology (10), chromatography for cell wall lipids and serotyping.  M. leprae does not grow on artificial media and one must rely on clinical findings and consistent histopathology for the diagnosis (6).

Culture of Brucella

Brucella is a Gram-coccobacillus.  Isolation from blood or tissues is done on bacterial media under aerobic or anaerobic condition.  The organism is fastidious, grows on enriched media such as trypticase soy agar supplemented with 5 percent sheep blood, brain heart infusion agar or chocolate agar under 8-10 percent CO2.Growth is slow may take 7 or more days, but it is recommended to hold cultures for 3 weeks.  Additional flora is sometimes suppressed by added bacitracin,polymyxin inhibitors.  On positive plates small non-hemolytic colonies appear.  Biochemical activity such as catalase, urease, oxidase, H2S production and sensitivity to basic fuchsin aid in the correct identification (37).

Culture of Listeria

Listeria is a Gram+ coccobacillus.  It can be grown on sheep blood agar where it shows β hemolytic action, and brain heart infusion agar with added glucose.  It can break down esculin by its β glucosidase enzyme and exhibits phospholipase activity as virulence factor.  Those properties are utilized in a selective chromogenic medium where glucosidase activity on a chromogenic substrate is observed.  Within 24-48 hr blue turquoise colonies appear and phospholipase action generates a white precipitate around the colonies (37).

Culture of Fungi

Most clinically important fungi can be isolated on Sabouraud’s dextrose agar with specific modifications (Emmons) that favor growth of common clinical species. Cultures are usually positive within 3-7 days, though certain fungi may take weeks to grow. Such is Histoplasma capsulatum a fastidious dimorphic fungus that grows in the yeast form in the lungs. Granulomas can be isolated by bronchoscopic biopsy. The fungus can be grown on brain heart infusion agar with added gentamicin, vancomycin chloramphenicol antibiotics to prevent the growth of contaminating bacteria. At 30-37oC it grows slowly (2-4 wks) as budding yeasts. Nocardia and Actinomyces will grow on routine media taking up to 2 weeks to yield a positive culture.

Stains

Multiple differential stains are available to the pathologist for identification of potential infectious causes of granuloma. The tissue sample has to be properly fixed to preserve the cellular and acellular elements of the biopsied material. The paraffin-embedded tissue is sliced to 5-6µm thick sections and stained by a variety of stains that reveal the various cellular and tissue elements.

Haematoxylin-Eosin (H&E) is the most widely used stain for the microscopic diagnosis of granuloma etiology. The lesions are usually well-delineated from the surrounding tissue. The stained slide will show cellular nuclei stained purple and cytoplasm stained light rose or red. Such stain can distinguish by morphology lymphocytes, macrophages, epithelioid cells, granulocytes and fibroblasts. Within the granuloma the characteristic palissading arrangement of the epithelioid cells interspersed with lymphocytes or the peripheral lymphoid cells is seen. Central caseous necrosis, giant cells with numerous nuclei provide clues for the architecture and /or the etiology of the lesion. Eosinophils and mast cells present in helminthic granulomas may be degranulated and need special tissue stains for identification (16).Some microorganisms can be seen and identified within the granuloma by the H&E stain.

Dominici stain is used for the identification of tissue eosinophils which stains the granules bright red. A more specific identification uses immunofluorescence microscopy. A primary antibody prepared against the cationic major basic protein (MBP) within the eosinophilic granules binds to the protein. Then a fluoresceinated secondary antibody that recognizes the primary antibody is applied. After the proper washes and controls the preparation is identified under the microscope where the granules show bright green fluorescence.

Acidified Toluidin blue stain is used for the identification of tissue mast cells. The sulfated acid mucopolysaccharides within the granules stain red to purple. A more specific immunohistochemistry method uses a specific antibody prepared to the tryptase enzyme within the granules of the mast cells. The bound primary antibody couples with the secondary antibody-biotin-streptavidin-horseradish peroxidase complex which stains the granules reddish brown. Non-specific esterase stain is used for the detection of tissue macrophages. The substrates used are naphtyl acetate or butyrate. The cytoplasmic enzyme stains red.

Immunohistochemistry, Immunofluorescence

Advances in the identification of specific surface markers of T and B lymphocyte and the different T lymphocyte phenotypes made the preparation of specific antibodies possible. Presently an array of commercially available antibodies can identify T lymphocytes (anti- CD3 marker), T helper lymphocytes (anti-CD4 marker), cytotoxic T cells (anti-CD8 marker), B lymphocytes (anti-CD20 marker), and macrophages(anti-CD68 marker).  Application of such antisera to the granuloma tissue with the avidin-biotin-horseradish peroxidase complex can reveal the exact identity and location of the granuloma cells.An elegant refinement uses double immunofluoresence for further analysis of the T helper lymphocyte subset. The first antibody tagged with one fluorochrome will identify the CD4+ T cell while a second antibody specific for a certain cytokine(IFNγ,IL-4,IL-10 etc) with a different fluorochrome tag penetrates the cell membrane rendered permeable by a chemical and will stain the intracellular cytokine. The intracellular cytokine content either IFNγ or IL-4 will identify the T helper1 (Th1)or T helper 2 (Th2) phenotypes respectively of the CD4+ T cell,which in turn characterizes the granuloma and predicts its efficacy in the elimination of an intracellular invader such as M. tuberculosis.

Application to the granuloma of a specific stain for the identification of an invader can provide the final evidence for the etiology of the lesion. This sometimes is fraught with difficulties. A very efficient granuloma should show a paucity of the invader having had eliminated the majority of it by cellular mirobiocidal activity. In fact scrutiny of the biopsied material from patients with confirmed clinical TB reveals only a low percentage of acid fast bacilli (AFB) positive lesions.  Nevertheless the gold standard for the diagnosis of TB is the presence of AFB in the granuloma tissue. The standard stain is the Ziehl-Neelsen acid fast stain.  An alternative stain for M. tuberculosis detection is the auramine-rhodamine fluorescent stain that has affinity for the mycolic acid cell wall component of the bacilli.  In a dark field bacilli are bright yellow.  Such stain enhances the sensitivity of detection(11).However the presence of AFB within the granulomas does not prove that the pathogen is M. tuberculosis, because acid-fast M. avium or other mycobacteria may also be present.

Treponema pallidum is also difficult to detect by regular stains.  Needle-aspirated regional lymph node tissue can be stained with fluorescein tagged anti-treponema antibody and under dark field the stained bacilli fluoresce bright green.

Localization by immunohistochemistry within sarcoid-like granuloma macrophages of inhaled pigeon antigen helps to confirm the diagnosis of hypersensitivity pneumonitis. Fungi are detected in tissues with Gomori’s methenamine silver stain that stains the cell wall brown to black.  The procedure may generate artifacts, therefore the morphology of the fungus has to be ascertained.  The periodic acid Schiff (PAS) reagent stains red the cell wall of Candida.  The Calcofluor white (fluorescent) staining may also be used to stain fungi. Specialized stains such as the Fontana-Masson to detect melanin in fungi and the mucicarmine stain for cryptococcal cell-wall polysaccharide may further assist in determining the fungal etiology. Despite the use of specific stains, in many instances organisms due to their paucity in the histopathologic specimen can not be demonstrated and cultures are needed for identification.

Stains for Non-Cellular Tissue Elements

Collagen appears in many granulomas especially at the healing phase of the lesion. The routinely used stain is Mallory’s trichrome which stains the collagen fibers blue, elastin fibrils pink or yellow and muscle fibers red.

The periodic acid Schiff (PAS) reagent stains glycogen as well as mucopolysaccharides in the tissues.

Molecular Methods

Staining of tissue sections and culture of viable pathogens can provide the most definitive diagnosis for the etiology of the granuloma. As mentioned in the previous sections the paucity of the pathogen such as AFB and low sensitivity of the staining method can prevent the correct diagnosis. Though by means of laser capture microdissection granulomas can be obtained free from the surrounding tissue (53) growth of pathogens (M. tuberculosis, fungi) may take weeks, delaying diagnosis.  The advent of molecular methods especially the polymerase chain reaction (PCR) as a diagnostic tool made the detection of uncultivable organisms possible. The standard, nested, or real time (RT) PCR enjoy wide-spread use because the laboratory can utilize for the assay the formalin-fixed, paraffin-embedded histological sections.  The sensitivity and speed of detecting a specific message for a pathogen may surpass that of a culture because result is obtained within 24 hr.  However, some of these assays are not standardized and caution must be used in the interpretation because false-positive results may occur.(13,27,38,44,48,53). Currently commercial kits with primer probes specific for pathogen DNA are available which greatly amplify the sensitivity of the detection. For M. tuberculosis diagnosis the insertion sequence (IS) 6110 is routinely used with great success. Compared with the efficacy of the Ziehl-Neelsen staining or culture, laboratories report double or triple sensitivity and specificity in detection.  Another commercial kit (Genprobe) for Mycobacterium tuberculosis detection targets the 16S rRNA genes with sensitivity and specificity of >95% in smear positive cases.  The usefulness of the assay in extra-pulmonary TB is unclear, particularly when histopathologic stains are negative.  The great advantage of the PCR assay is its applicability to detecting the DNA of any bacilli or fungi.  However it is labor-intensive because for each pathogen a specific probe has to be prepared or obtained. A more sophisticated assay uses the DNA microarray method in which multiple DNA probes with known identities are fixed on a glass surface and molecular hybridization is carried out with the pathogen DNA (36,45).This assay can simultaneously detect hundreds or thousands of genes.  Such approach is very helpful in the differential diagnosis when more than one pathogen has to be considered.  Low density microarray can simultaneously detect Mycobacterium spp, Yersinia spp, Bartonella and other pathogens(45).Microarray has also been applied to the simultaneous differentiation of Mycobacteria species.  Using the DNA gyrase B subunit (gyrB) genes as a probeMycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare and other species could be identified (23).

Use of Specific Antibodies and T Cell Function Assays

Detection of specific antibodies remains a cornerstone of diagnosis of many granulomatous diseases of infectious, or unknown etiology. Anti-neutrophil cytoplasmic antibodies may be the only clue to granulomatous vasculitis(46), and fastidious, slow-growing pathogens are often only suspected but diagnosis is confirmed when specific antibodies are positive. This may be particularly important for infections due to Coxiella, Brucella and Bartonella, among others. Fluorescence antibody techniques are becoming more commonly used for specific pathogens, though are not generally available for organisms causing granulomatous infections. Overall, the non-culture serological methods of diagnosis may be useful when traditional approaches fail to discern the cause of a granulomatous lesion and will likely increase in use as more commercial tests become available.  Examination of peripheral blood or bronchial T cell responsiveness specific for a pathogen can provide evidence for the immune etiology of the granulomatous disease.  Thus, in vitro T cell proliferation ,or cytokine (IFNγ,IL-4, etc) production to a soluble antigen of a pathogen,or detection of intracellular cytokines by immunofluorescence in laser capture microdissection- isolated granulomas serve as important corollaries in the diagnosis. Finally, the time-honored and simple intradermal test using PPD for TB detection, or fungal antigens can indicate either a past exposure to the pathogens or an ongoing infection. Either way it reinforces the infectious etiology of the granulomatous disease.

Histopathology

The histologic appearance in stained sections of a particular granulomatous lesion may provide information as to its etiology. The morphology (compact organized, diffuse, encased in fibrous capsule) and the cellular composition of the lesion are helpful guidelines(18).First,the immune or foreign body characteristics have to be established. The latter can be diagnosed with some assurance because the irritant (talc, quartz, silicon, graphite, suture) is present within the granuloma. As mentioned in previous sections neither the presence of epithelioid nor the type of giant cells can differentiate between the two types of granulomas. The presence within the granulomas of CD4+ or CD8+ T lymphocytes is indicative of an immune etiology. This is the case in sarcoidosis(4) or Crohn’s disease(25,59).

Histopathological differentiation of the various granulomatous diseases is a challenging task because often neither the morphology nor the cellular composition can furnish a clue. The M. tuberculosis induced tubercle, the beryllium metal-induced pulmonary granuloma (39,58) or the sarcoid lesion (4,18,30) all contain epitheliod cells and T lymphocytes. The central caseating necrosis associated with granulomas is characteristic of tuberculous, but not the beryllium-induced, sarcoid or Crohn’s disease lesions (Table 3).  An additional difficulty for the pathologist is the morphological spectrum seen in TB or leprosy (2). In TB tissue responses range from sheets of foamy macrophages packed with acid fast-staining bacilli to hematogeneously spread 1-3 mm size miliary granulomas in immunocompromised humans, to caseting or non-caseating organized epithelioid garnulomas (51). Necrotizing granulomas are often indicative of vasculitic lesions. In the necrobiotic (collagenolytic) granulomas the cellular infiltrate contains also neutrophils and eosinophils (35,55). The presence of associated pathologic findings can also be useful, such as the eosinophilic infiltrate and/or eosinophil cationic protein in the serum in Churg-Strauss disease. Examination of stained slides does not only aid in the correct diagnosis of the disease, but can be a valuable tool in retrospective analyses of archival material. Detection in hundreds of slides of AFB of proven TB cases can establish a correlation between the presence of AFB and active or dormant infection.  A multivariate analysis of the presence of epithelioid granulomas with subsequent surgical bowel resection revealed that in Crohn’s disease the frequency of granuloma presence may indicate a more aggressive disease process (25).

The usual dilemma, however, is what to make of granulomatous inflammation when none of the above clues is present, and repeat biopsies and cultures may be necessary to arrive at a final diagnosis.

Clinical Clues in Assessing Etiology

The most useful aspect of arriving at a diagnosis is the history of the illness and sometimes the occupation of the patient (berylliosis). This can often give clues for infectious or non-infectious causes regarding exposures and course of illness. Indeed, most granulomatous infections have chronic, indolent presentations. Exposure to tuberculosis or a positive PPD skin test result may lead to empiric therapy if the clinical suspicion is high. Of the fungal etiologies, the endemic fungi have geographic restrictions (histoplasmosis) that are helpful in narrowing the differential. Candida is associated with intravenous drug use and Aspergillus with neutropenia. Brucellosis, Q-fever and cat-scratch disease are zoonoses that have relatively specific animal exposures associated with them. Nocardia is usually related to prior steroid use, and Actinomycosis will frequently present with a draining sinus tract in the area of infection. Specific organ involvement is also useful, due to unique disease manifestations for that organ system. Renal disease might point one towards vasculitis, while lung disease would suggest a work-up for sarcoidosis. Table 4 provides serologic testing and other recommendations for diagnosis of the various infectious causes of granulomas.

Other studies that may assist in diagnosis include more sophisticated imaging, such as computed tomography (CT) of the chest to better characterize lesions seen on chest x-ray and detect lymphadenopathy that is not apparent on clinical examination. This usually leads to biopsy of suspicious lesions that eventually makes the diagnosis.

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Tables

Table 1: A Non-inclusive List of Granulomatous Diseases and Their Causative Agents.

Inducers	Diseases
Bacteria	Brucellosis Syphilis Bartonellosis Lymphogranuloma venereum Tuberculosis Leprosy
Fungi	Histoplasmosis Coccidioidomycosis Blastomycosis
Protozoa	Leishmaniasis
Helminths	Filariasis Trichinosis Schistosomiasis
Organic dust	Hypersensitivity pneumonitis
Metals	Berylliosis Zirconium granulomas Sarcoidosis Crohn’s disease
Unknown	Primary Biliary cirrhosis Wegener’s granulomatosis Granuloma annulare
Inanimate agents	Silicosis Asbestosis Granulomatous inflammations to inanimate    Surgical sutures    Silicone    Talc    Tatoo pigment    Keratin    Aspirated material
Malignancy	Malignant tumor-associated granulomas    Hodgkin’s disease    T cell lymphomas    Seminoma of the testis    Large cell lung carcinoma

Table 2: Causes of Suppurative (necrotic) or Calcified Granulomas

Necrosis or suppuration		Calcification
T. pallidum		M. tuberculosis
B. melitensis		B. melitensis
Y. pestis, Y. enterocolica		H. capsulatum
L. monocytogenes		T. spiralis
Chlamydia trachomatis		Cysticercosis
Bartonella henselae
C. albicans
A. fumigatus
Blastomycis dermatitidis
Coccidioides immitis
N. asteroides
Wegener’s disease
Granuloma annulare

Table 3:  Causes of Caseating versus Non-caseating Granulomas

Non caseating granulomas		Caseating granulomas
M. leprae  (tuberculoid form)		M. tuberculosis (cavitation)
Schistosoma mansoni eggs		T. pallidum
Crohns disease		H. capsulatum
Beryllium, Zirconium metals		B. melitensis
Sarcoidosis		C. neoformans
Hypersensitivity pneumonitis          -Bird antigens          -Actinomycetes

Table 4: Pathologic Findings and Supportive Diagnostic Tests for Granuloma Inciting Infections

Organism	Pathologic Findings	Supportive studies
Aspergillus	Invasive pulmonary necrotizing granulomas, hyphae, aspergilloma	Galactomannin (Aspergillus antigen)
Bartonella sp.	Necrotizing liver granulomas	Serologies, Blood cultures held for 4 weeks
Blastomyces dermatitidis	Pulmonary granulomas, abscesses, fibrosis,  broad based budding yeast	Serologies, Urine antigen
Brucella sp.	Liver, spleen granulomas with central  necrosis, peripheral fibrosis, bacilli	Serologies
Candida sp.	Skin or other organs, abscesses granulomas with necrosis, yeast cells or hyphae	Routine culture
Coxiella burnetti	Granulomatous hepatitis, fibrosis, calcification	Serologies
Cryptococcus	Pulmonary abscesses, granulomas, fibrosis, yeast cells	Cryptococcal antigen
Cytomegalovirus	Granulomatous hepatitis, splenic granulomas	Viral culture, Serologies or PCR
Epstein Barr Virus	Weak association with skin granulomas,  granulomatous arteritis	Serologies or PCR
Filariasis	Subcutaneous granulomas, fibrosis and  calcification.  Granulomas block lymphatic  channels, adult worms	Peripheral blood smear
Histoplasma sp.	Lung granulomas, cavitation, yeast cells	Serologies by Complement Fixation or Immunodiffusion, urine or serum antigen
Leishmaniasis	Cutaneous granulomas with necrosis, lymph node granulomas, protozoa within macrophages	Culture and PCR
LGV or Chlamydia trachomatis	Inguinal lymphadenitis with granulomas	Culture and serology
Measles	Implicated in Crohn’s disease. Occurs as subacute granulomatous thyroiditis	Serologies
Mycobacterium sp.	Lung, liver granulomas with central caseous necrosis. Acid fast bacilli. Calcification	PPD, Quantiferon, AFB Culture
Nocardia sp.	Cutaneous or pulmonary granulomas and abscesses. Gram negative branching rods.	Modified Acid-Fast stain and culture
Schistosomiasis	Noncaseating granulomas in the liver,   intestines (S.mansoni ) or urinary bladder ( S. hematobium). Fibrosis. Bladder calcification. Worm eggs	Eosinophilia, identification of eggs in the urine, stool or tissue specimen.  Serologic testing
Syphilis	Noncaseating skin granulomas in secondary stage. Gumma formation in tertiary syphilis with large granulomas with necrotic center. Silver impregnation stain in tissues	RPR, FTA
Trichinellosis	Granuloma formation around the cysts with coiled larvae in the muscle	Muscle biopsy, eosinophilia
Tropheryma whippelii	Multisystemic caseating or noncaseating Granulomas, Gram positive rods	Small bowel biopsy, tissue for PCR

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