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 /+ 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 (NFB) 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 (TNF), interleukin-1 (IL-1), interleukin-12 (IL-12). Transmembrane TNF 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-1 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
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
Granulomatous Diseases
Authors: Dov L. Boros, Ph.D., Sanjay G. Revankar, M.D.
Table of Contents
Collapse All | Expand All
Function of the Granuloma
Morphology and Cellular
Composition of the
Granulomas
Mechanisms of Granuloma
Formation
Pathogenesis of the
Granulomas
Pathology of the Granuloma
Differential Diagnosis
Infectious Causes
Non-Infectious
Causes
Approach to
Determining
Etiology
Laboratory Diagnosis
Cultures
Stains
Immunohistochemistry,
Immunofluorescence
Stains for
Non-Cellular
Tissue Elements
Molecular Methods
Use of Specific
Antibodies and T
Cell Function
Assays
Histopathology
Clinical Clues in
Assessing Etiology
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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 / 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 TNF 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 IFN 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 TNF 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 TNF, IL-10 and the
regulatory TGF- 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 , TNF and TGF- 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 m 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-1, TNF, GM-CSF, IL-3, IL-6 cytokines and numerous chemokines both from the CCL (MCP-1, 2,
3, MIP-2, , ) 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, IFN, 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-, TNF-,
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-1, MIP-1, MIP-2 and RANTES that attract blood monocytes and lymphocytes to the site.
Concurrently, IL-1 and TNF 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 (TNF, IL-1,
IL-6, IL-10, IFN 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 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
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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 IFN 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 (TNF), 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
TNF-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-, TNF, 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- 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-, TNF- 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- 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- 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, Whipples
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.
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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). Crohns 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 Wegeners granulomatosis, Churg-
Strauss disease, and Takayasus 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 patients 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 hemolytic action, and brain heart infusion
agar with added glucose. It can break down esculin by its 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
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Most clinically important fungi can be isolated on Sabourauds 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 5-6m 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(IFN,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 IFN 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 Gomoris 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 Mallorys 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
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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 (IFN,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 Crohns 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 Crohns 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 Crohns 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
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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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