Distribution of Extracellular Matrices, Matrix Receptors, and Transforming Growth Factor-β1 in Human and Experimental Lung Granulomatous Inflammation

Distribution of Extracellular Matrices, Matrix Receptors, and Transforming Growth Factor-,Bl in Human and Experimental Lung Granulomatous Inflammation JESSE ROMAN, MD,* YOUNG-JUNE JEaN, MD,* RAFAEL L. PEREZ, MD*

ABSTRACT: Aberrant deposition of extracellular matrices (ECMs) may affect lung inflammation by influencing cell adhesion, migration, and activation. Little is known about the expression of ECMs in lungs with granulomatous inflammation. Therefore, the authors investigated the distribution of ECMs, matrix receptors of the integrin family, and transforming growth factor-beta 1 (TGF-,81) in lungs from patients with pulmonary sarcoidosis and animals with experimental granulomatosis. Immunohistochemistry revealed increased deposition of type I collagen and fibronectin in human lung granulomas when compared with healthy human lungs. Procollagen type I and cellular fibronectin also were increased, suggesting local synthesis of ECM in sarcoid granulomas. These findings were accompanied by increased staining for fibronectin (0:5,81) and collagen (0:2,81) integrin receptors. The matrix-inducing cytokine TGF-,81 was codistributed with the aforementioned molecules in the granulomas, whereas no significant staining for TGF-,81 was found in healthy lungs. Similar to sarcoid lungs, analysis of lung sections obtained from a murine model of granuloma formation revealed increased expression of fibroFrom the *Department of Medicine, Atlanta Veterans Affairs Medical Center, Atlanta, Georgia, and the t Department of Pathology, Emory University School of Medicine, Atlanta, Georgia. Dr. Young-June Jeon was supported by institutional funds from Keimyung University School of Medicine, Korea. Dr. R. L. Perez was supported in part by The Carlyle Fraser H eart Center of Crawford Long Hospital, Atlanta. This research was supported in part by research grants from the American Lung Association, American Heart Association, and HL5J639-0J from the National Institutes of Health (J. Roman). J . Roman is a recipient of a Minority Medical Faculty Development Award from the Robert Wood Johnson Foundation, New Jersey. Correspondence: Jesse Roman, MD, Department of Medicine (I J1), Veterans Affairs Medical Center, 1670 Clairmont Road, Decatur, GA 30033.

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nectin, collagen, integrin receptors, and TGF-,81 within granulomas. Based on these observations, there is increased expression of ECM and matrix receptors in both human and experimental lung granulomas. Such alterations may influence the recruitment and activation of inflammatory cells and fibroblasts, promoting granuloma formation and remodeling of tissue by fibrosis. Activation of mononuclear cells resulting in production of TGF-,81 is likely to contribute to the changes described. KEY INDEXING TERMS: Granuloma; Extracellular matrix; Integrin; Transforming growth factor-,81; Sarcoidosis; Inflammation; Lung; Mycobacteria. [Am J Med Sci 1995; 309(3): 124-133.]

G

ranulomatous lung inflammation is characterized by the accumulation of macrophages and other inflammatory cells within the pulmonary interstitium followed by the formation of multiple central cores of large epithelioid cells surrounded by lymphocytes, multinucleated giant cells, macrophages, and fibrous connective tissue. 1 The granulomatous lesion is typical of diseases such as sarcoidosis, berylliosis, hypersensitivity pneumonitis, and tuberculosis. The mechanisms that lead to the organization of inflammatory cells into the characteristic granuloma are largely unknown, and the factors that perpetuate the inflammatory state or promote regression have not been identified. Several investigators demonstrated increased extracellular matrix (ECM) deposition in granulomatous and non granulomatous forms of lung injury.2-5 This, plus the ability ofECM components to promote chemotaxis, adhesion, and proliferation of inflammatory cells and fibroblasts in vitro, suggests a role for these factors in lung inflammation.s-9 Little is known about the ECM components involved in the granulomatous lung inflammation found in paMarch 1995 Volume 309 Number 3

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tients with sarcoidosis and the factors promoting their expression. In addition, the origin and function of these matrix components in granulomatous lung disease has not been-studi!l4 extensively. The receptors niediating cell-matrix interactions in granulomatous inflammation also are unknown. This study was performed to begin to identify the ECM molecules and receptors involved in the inflammatory response that characterizes granulomatous lung disease. We used immunohistochemical techniques to examine the distribution of collagen and fibronectin in formalin-fixed, paraffinembedded, open-lung biopsy sections obtained from patients with pulmonary sarcoidosis. We focused on these molecules because their expression is increased in other forms of lung injury, including pulmonary fibrosis and the adult respiratory distress syndrome.3,4 We also examined the tissue distribution of the collagen- and fibronectin-binding integrin receptors a2~1 and a5~1, respectively, which playa major role in determining cell responsiveness to these components. IO - 15 In addition, we examined the distribution of transforming growth factor-~l (TGF-~l), a cytokine known to stimulate the expression of ECM and integrin receptors in vitro which also has been implicated in lung disorders. 16-19 Finally, we compared our findings with those obtained in lung sections from animals with experimentally induced granulomatous inflammation. Based on our studies, human and experimental granulomatous lung inflammation are accompanied by increased expression and deposition of ECM components fibronectin and collagen. These molecules are likely to affect immune cell function because mononuclear cells within the granulomas expressed matrixbinding receptors of the integrin family. The expression of the matrix-inducing cytokine TGF-~l also was increased in granulomatous lungs, suggesting a functional role. Based on our observations, similar mechanisms are involved in both human and experimental pulmonary granulomatosis. In addition, these observations help explain the link between the early inflammatory events that induce granuloma formation and the subsequent events that sometimes lead to progression toward pulmonary fibrosis. Finally, based on the data presented, the experimental model may be an adequate model in which to study the mechanisms responsible for granuloma formation. Materials and Methods Human Tissues. Paraffin-embedded open lung biopsy

specimens from two patients with sarcoidosis were obtained from the tissue banks of the Department of Patho logy at our institution. Paraffin -embedded normal lung tissue was obtained from the contralateral lung of a single lung transplant donor (provided by Dr. Samuel M. Aguayo, Department of Medicine, Veterans Affairs Medical Center/Emory University, Atlanta, GA). Multiple tissue sections with a thickness of 6 JLm were prepared from each block. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

Antibodies. Fibronectin was detected with a polyclonal antibody (FN-1) raised in rabbits immunized with bovine plasma fibronectin, as described. 15 Immunoglobulin G was isolated by precipitation of serum with octanoic acid followed by affinity chromatography on a fibronectin -Sepharose column. Immunofluorescence staining of human and mouse fibroblasts with FN-1 revealed reactivity against human and mouse fibronectin (not shown). The specificity of this antibody toward fibronectin was tested in cell adhesion assays and by enzyme-linked immunosorbent assays and Western Blot analysis (not shown). The polyclonal anti-fibronectin antibody Ab 1.5 also was used. 20 Antibodies to Type I collagen (polyclonal antibody AB747), to human type I pro collagen (monoclonal antibody 1913), and to cellular fibronectin (monoclonal antibody 1940 directed to the EIIIA domain) were purchased from Chemicon International, Inc., Temecula, CA. A polyclonal synthetic peptide antibody directed to the cytoplasmic domain of the a5 subunit of the a5~1 fibronectin receptor has been described. 15 The monoclonal antibody P1D6 also was used to detect the a5 subunit (Telios Pharmaceuticals, Inc., San Diego, CA). A monoclonal antibody to the a2 subunit of the integrin collagen receptor a2~1, clone P1E6, A042 was purchased from Telios Pharmaceuticals, Inc., San Diego, CA. Transforming growth factor-~l was detected with one of two antibodies, Rb4 (generously donated by Jim Dasch, Celtrix Pharmaceuticals, Santa Clara, CA) and A1/30 (also known as CCl-30; generously donated by L. E. Ellingsworth, Celtrix Pharmaceuticals). Both antibodies were raised against the same TGF~1 aminoterminal synthetic peptide, but are believed to contain different populations of epitope-specific immunoglobulins. 21 Although the two rabbit antisera have similar reactivities in Western Blots, immunocytologic analysis suggests they recognize the mature sequence of TGF-~l in different contexts. Rb4 (like LC-1-30 21 ) stains TGF-~l in an intracellular form,22 and A1/30 stains an ECM -associated form. 23 Rb4 stained human tissue sections but failed to stain mouse sections that were stained with A1/30. This may be due to differences in the antigen used to develop the antibodies versus problems created by the different fixation protocols. Immunohistochemistry. Human and murine tissues fixed in 10% phosphate-buffered formalin were embedded in low-melting paraffin. Sections 6 JLm thick were deparaffinized and treated with 1 % hydrogen peroxide in methanol to quench the activity of the endogenous peroxidase. The sections were rehydrated in phosphate-buffered saline (PBS, pH 7.2), incubated in 0.3% Triton in PBS with bovine serum albumin for 20 minutes in humidifying chamber and then rinsed with PBS. Nonspecific protein binding was blocked with 1.5% goat serum and Avidin/Biotin blocking reagent (Biogenex Laboratories, San Ramon, CA). The primary antibodies were diluted with 1 % bovine serum albumin

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in PBS with bovine serum albumin. The sections were incubated with the optimal dilution of the primary antibodies for 1 hour in a humidifying chamber at room temperature. The sections were rinsed with PBS and incubated with biotinylated goat anti-rabbit or horse anti-mouse immunoglobulin (Vector Laboratories, Burlingame, CA) for 30 minutes, washed for 20 minutes, incubated with avidin-biotin-peroxidase complex for 30 minutes, washed for 20 minutes, and visualized with 3,3'-diamino-benzidine substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's recommendations. The sections were counterstained with hematoxylin for 1 minute. Controls included lung sections stained with non-immune polyclonal and monoclonal immunoglobulin G and in the absence of primary or secondary antibody. Induction of Pulmonary Granulomas in Mice. Eightweek-old female ICR mice (Charles Rivers, Raleigh, NC) were acclimated to the animal facilities for 1 week before induction of pulmonary granulomas. Trehalose6,6'-dimycolate (TDM) obtained by petroleum ether extraction from Mycobacterium smegmatis24 was a gift of Dr. Kuni Takayama, Madison, WI. Fifty-two microliters of solubilized TDM at a concentration of 1 JLg/JLL in hexane:ethanol (ratio of 9: 1) were placed into a glass pestle, and the solvent was evaporated under a stream of nitrogen gas. The dry TDM was ground into 50 JLL of the light mineral oil VR6. An oil-in-water emulsion was prepared by homogenization with 1.25 mL of the vehicle 0.2% Tween 80 in PBS. One tenth milliliter of emulsion prepared with TDM (4 JLg per mouse) was injected intravenously via tail vein to produce lung granulomas. Animals given oil emulsion without TDM or vehicle without TDM served as control subjects. Granuloma formation is observed as early as 24 hours after TDM administration. The number of granulomas peaks at 48-72 hours, decreasing thereafter, and are absent by day 16. 25 Lungs were harvested 2 days after TDM injection, when granulomas are most numerous. Mice were anesthetized with 4 mg of sodium pentobarbital and exsanguinated by severing the abdominal aorta to prevent passive pulmonary congestion. The lungs were cannulated in situ, removed en bloc, fixed in inflation with 10% buffered formalin at 15 em water pressure, and processed for immunohistochemistry as discussed earlier. Sections obtained from four control and four experimental animals were examined. Results Distribution of Fibronectin and Collagen in Sarcoid Lung. Human healthy and sarcoid lungs incubated with

non-immune antibodies did not reveal specific staining (Figures 1A and 1B, respectively). Immunohistochemical analysis of sarcoid lung sections with anti -collagen and anti-fibronectin antibodies revealed a remarkable increase in these ECM components (Figure 1) as compared with healthy lungs (not shown). Staining for type

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I collagen was prominent at the periphery of the granulomas, sparing their central cellular core (Figure lC). However, fibronectin was present in cells and in stromal elements within and surrounding sarcoid granulomas (Figure ID). Both FN -1 (Figure 1D) and Ab 1.5 (not shown) revealed identical staining patterns for fibronectin. To investigate whether the increase in collagen and fibronectin staining observed in sarcoid lungs was due to increased expression or reorganization of these molecules, we examined the distribution of procollagen type I and cellular fibronectin. Distribution of these ECM components detected by immunohistochemical methods correlates well with synthesis. 3,4,26 Procollagen type I is the monomeric soluble precursor of collagen type I which, along with collagen type III, composes most of the collagen pool in the pulmonary interstitium. 27 Procollagen is increased in fibrotic lungs, and its expression correlates with the presence of "activated matrix-forming" fibroblasts typically found in remodeling tissues. 3,4,26 The fibronectin detected with monoclonal antibody 1940 is a fibronectin variant that contains an extra type III repeat resulting from variable splicing of a single fibronectin gene. 28 This fibronectin variant will be referred to as "cellular fibronectin" for the purpose of our discussion. Normal adult tissues express little cellular fibronectin, mainly localized to major vessels, whereas injured tissues express much greater amounts of cellular fibronectin. 29 Immunostaining for procollagen type I was concentrated on cells within the granuloma and on fibroblasts scattered throughout the collagen fibrils between granulomas (Figure IE). Similarly, staining for cellular fibronectin was seen in the center of the granulomas, with prominent uptake in multinucleated giant cells as well as macrophages and surrounding fibroblasts (Figure IF). Staining for fibronectin, including cellular fibronectin, was observed in macrophages from adjacent alveoli in healthy and granulomatous lungs. This is not surprising, because alveolar macrophages constitutively express fibronectin. 20 No procollagen type I and little cellular fibronectin, mainly within vessels, were detected in healthy lungs, as previously described by others (not shown).3,4,26 Distribution of Fibronectin and Collagen Receptors in Sarcoid Lung. The biologic effects of ECMs on cell

behavior are mediated via cell surface molecules that include members of the integrin family. Integrins are transmembrane heterodimeric glycoproteins that mediate cell-cell and cell-matrix interactions. All integrins are composed of one a and one (3 subunit that bind ligand extracellularly and interact with the cytoskeleton intracellularly.l0,13,14,30-32 We examined the distribution of two integrin subunits, the a5 subunit of the a5(31 fibronectin receptor that mediates most of the cellular effects of fibronectin (Figure 2A and C), and the a2 subunit of the a2(31 collagen receptor (Figure 2B and D). We found that mononuclear and mulMarch 1995 Volume 309 Number 3

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Figure 1. Distribution of fibronectin and collagen in human sarcoid lung. Control human lung (A) and human sarcoid lung (B) tissues had no staining with non-immune antibodies. Type I collagen was deposited at the periphery of granulomas (C) . In contrast, fibronectin (detected with FN-1) was present in cellular and stromal constituents within and sur· rounding the granulomas (D). Both procollagen type I (E) and cellular fibronectin (F) were distributed within multinucleated giant cells, macrophages, and surrounding fibroblasts. (A and B magnified X 100; C-F magnified X 200) .

tinucleated giant cells within the granulomas, macrophages, and fibroblasts enmeshed in fibrotic areas stained specifically for the integrin fibronectin and collagen receptors. Identical results were obtained for a5 staining with the monoclonal antibody PID6 (not shown). Staining for these receptor subunits appeared to be increased when compared with healthy lungs. As previously reported in healthy lungs, a2 staining was detected in bronchial epithelium, large vessel endothelium, and bronchial smooth muscle (not shown). The a5 subunit was detected in alveolar septae and in vessels, but not in bronchial epithelium (not shown).13,30,33 Distribution of Transforming Growth Factor-,8i in Sarcoid Lung Tissue. Binding of TGF-,81 to specific cell

membrane receptors induces the expression of both ECMs and their receptors in vitro. 18,19 To determine whether TGF-,81 was expressed in granulomatous lungs, we stained lung sections with an antibody that detects TGF-,81. We observed that TGF-,81 was prominent in sarcoid lungs codistributing with procollagen, THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

cellular fibronectin, and thea2 and a5 integrin subunits (Figures 3B and 3C). Macrophages were intensely stained for TGF -,81. Transforming growth factor-,81 also was prominent in giant cells within the center of granulomas and on fibroblasts in fibrotic areas and bronchial epithelium. In contrast to sarcoid lungs, TGF-,81 was not detected in healthy lungs (Figure 3A). Distribution of Extracellular Matrices, Matrix Receptors, and Transforming Growth Factor-,8i in a Murine Model of Lung Granulomatous Inflammation. To de-

termine whether experimental granulomatosis also was associated with increased matrix gene expression and TGF-,81 production, we analyzed lung sections from ICR mice 48 hours after the administration of TDM. Trehalose-6,6'-dimycolate is a glycolipid purified from the cell walls of mycobacteria that produces pulmonary granulomas after intravenous administration. 25,34 Lung granulomas produced by TDM in mice differed morphologically from pulmonary granulomas in human sarcoidosis. In contrast to sarcoid granulomas, TDMinduced granulomas were formed by less organized but

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Figure 2. Distribution of integrin subunits in human sarcoid lung. Intense staining for the a5 subunit of the a5{31 fibronectin receptor was localized in cells within the centers of granulomas as well as in fibroblasts surrounding the lesions (A and B). The a2 subunit of the a2{31 collagen receptor also was localized centrally (C and D). with additional staining seen in fibroblasts around the granulomas (A and C magnified X 100; Band D magnified X 200).

discrete aggregates of mononuclear cells that did not contain giant cells and were not surrounded by fibrotic stroma. These granulomas were uniform in appearance presumably because they were all induced simultaneously by intravascular administration of TDM.

There was no specific staining in control or TDMtreated lungs incubated with non-immune antisera (Figures 4A and 4B). Type I procollagen was detected on cells throughout the TDM-induced granulomas (Figure 4C) but not in control lungs (not shown). Fi-

Figure 3. Distribution of transforming growth factor-{31 (TGF-{31) in human sarcoid lung. Control human lung had no immunoreactivity for TGF-{31 when stained with Rb4 (A). In sarcoid lung, TGF-{31 was prominent in the center of granulomas, giant cells, fibroblasts, and in bronchial epithelium (B and C). (A and B magnified X 100; C magnified X 200).

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Figure 4. Distribution of extracellular matrices, matrix receptors, and transforming growth factor-In in murine model of -granuloma formation. Control (A) and trehalose-6,6' -dimycolate-treated (B-E) murine lungs had no immunoreactivity for non-immune sera. There was strong, diffuse staining throughout granulomas for procollagen type I (C), fibronectin (D), fibronectin receptor, a5{31 (E), and transforming growth factor·{31 (F). A 1/30 was used to detect transforming growth factor-{3l. The FN·l antibody was used for detection of fibronectin. (A and B magnified X 100; C-F magnified X 400).

bronectin was concentrated at the rim and between apposed cells extending into the granulomas in TDMtreated mouse lungs (Figure 4D). As in humans, in control murine lungs, fibronectin was detected predominantly in vessels and alveolar septae, and faint staining was seen in alveolar macrophages (not shown). Cellular fibronectin was not examined because the antibody available to this molecule was developed in mice. The fibronectin receptor a5 subunit was detected in THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

mononuclear cells throughout the TDM-induced granulomas and on alveolar macrophages (Figure 4E). In control lungs, a5 was detected in vessels, alveolar septae, and alveolar macrophages (not shown). Similar to the sarcoid graJ;lulomas, TGF-/31 was expressed prominently and was found co-distributing with procollagen, fibronectin, and integrin receptors (Figure 4F), whereas no TGF-/31 was detected in control lungs (not shown).

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Discussion Extracellular Matrices in Pulmonary Granulomatosis.

Granulomas that occur in a wide range of diseases of known and unknown etiologies range in morphologic complexity from discrete aggregates of mononuclear cells to complex accumulations or epithelioid cells surrounded by lymphocytes and fibrosis. 35 Although past work has focused on various cellular aspects of granuloma formation, little is known about cell interactions with surrounding matrix proteins and mediators in granuloma formation. To begin to understand how the ECM may affect granuloma formation and lung inflammation, we examined the morphologic relations between matrix proteins, their specific cell-integrin receptors, and the matrix-inducing cytokine TGF-~l in human lungs with granulomatous inflammation caused by sarcoidosis. It must be emphasized that the findings described in this article have been obtained from examination of tissue samples from two patients with sarcoidosis. More cases could not be obtained for study because open lung biopsy is no longer performed to diagnose this condition. Instead, patients undergo fiberoptic bronchoscopy and transbronchiallung biopsy, which provides limited quantities of small tissue samples. However, parallel findings have been reported previously in other patients with sarcoidosis using different reagents directed at fibronectin, a5~1, and TGF-~1.36 Therefore, that report and the fact that all granulomas examined in multiple sections had the same characteristic findings suggest that our observations are likely typical of sarcoidosis. We compared our findings in human tissue with those obtained in experimental animals with TDMinduced granulomas. We do not equate TDM-induced lung granulomatosis with sarcoidosis, but we use the model to examine the mechanisms involved in granuloma formation. The close distributional relation between ECM, ECM receptors, and the cytokine TGF~1 in granulomas of clinical and experimental pulmonary granulomatosis suggests common pathways involved in granulomatous inflammation of the lung irrespective of host or causative agent. We found that type I collagen was distributed in a circumferential pattern around sarcoid granulomas sometimes extending in between cells forming the center of the lesions. Fibronectin was found in the periphery as well as the center of granulomas. Some cells at a distance from the granulomas appear to show staining with these and other antibodies tested. This is explained by the fact that many cells express the molecules tested even in normal states. For example, fibronectin is synthesized by many cell types, including endothelial cells and alveolar macrophages. In addition, certain cells may not synthesize or assemble fibronectin into insoluble matrices but may stain for this molecule because they bind to fibronectin via fibronectin receptors. Because these molecules are constitutively ex130

pressed in some cells, it is not surprising that staining was detected in cells within and outside the granulomas as well as in cells in healthy lungs. A similar distribution for collagen and fibronectin was described in a model of Schistosome egg granulomatosis. 37 Others also demonstrated increased deposition of collagen, fibronectin, and fibrin in sarcoidosis and experimental granulomas, but the sources of these matrix components were not determined. 5,38-42 We observed that sarcoid granulomas expressed both procollagen type I and cellular fibronectin; TDM-induced granulomas also expressed procollagen type 1. Based on these observations, one source of the ECM proteins that surround cells composing granulomas may be the granulomas themselves. The differences in the staining pattern between collagen and procollagen type I in well-formed sarcoid granulomas was intriguing. Collagen was detected predominantly at the periphery of granulomas, whereas procollagen was present within the granulomas. This demonstrates that procollagen staining is not a useful indicator for where collagen will eventually be deposited. More important, this is consistent with the idea that cells forming the core of granulomas (such as monocytes and macrophages) are capable of synthesizing and secreting soluble matrix components but cannot incorporate them into insoluble matrix fibers. 43 Fibroblasts, however, (which are localized at the periphery of the granuloma core), express matrix components and assemble them into their matrix. 15 Detection of procollagen in cells of monocytic/ phagocytic lineage is intriguing for yet another aspect. Although mononuclear cells are not usually considered collagen-producing cells, there are some instances in which this may occur. Several studies reported the transformation ofHLA-DR monocytes into neo-fibroblasts under conditions associated with tissue remodeling, such as in osteomyeloslerosis and tumor encapsulation. 44 ,45 This transformation appears dependent on the presence of T -lymphocytes and is associated with the production of collagen. 46 Further work is needed to examine the potential implications of this process in granulomatous lung disease. Although the exact role of ECM in any form of lung disease is unknown, newly deposited matrices may provide provisional and permanent support for the accumulation of inflammatory cells and fibroblasts. 3,6,7,47 Adhesion to the matrix may regulate inflammatory cell function by inducing cytokine expression. 48-5o In addition, soluble fragments of collagen and fibronectin are chemotactic and promote inflammatory cell recruitment into injured tissues. 9 ,51 Matrix-Binding Integrins in Pulmonary Granulomato-

sis. Integrin receptors are important for cell binding to ECM proteins. Because this binding may alter cell behavior in ways that determine the type of inflammatory response,1O,13,30,52-54 granulomatous inflammation may result, in part, from specific integrin-ECM binding inMarch 1995 Volume 309 Number 3

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teractions. Immunostaining of human and experimentally induced pulmonary granulomas showed that cells within the granulomas, fibroblasts in fibrotic regions of the lung (seen only in sarcoid granulomatosis in our studies), and macrophages in the alveolar spaces were armed with collagen (a2,81) and fibronectin (a5,81) receptors. Although, we cannot determine whether the observed increase in staining for integrins is due to increased number of cells versus increased cellular expression, our observations suggest that cells within and surrounding granulomas not only produce ECM, but once these components are released, integrin-armed cells can bind to the newly formed ECM. Integrins may mediate ECM-induction of cytokine expression. For example, collagen binding to the integrin a2,81 induces production of the proinflammatory cytokine interleukin -1,8 by cultured peripheral blood mononuclear cells. This effect is potentiated by ligand binding to the fibronectin receptor a5,81. 49 ,50 We would like to highlight the observation that staining for a2 did not co-distribute with collagen but with procollagen. This may be explained by the presence of very few cells in the fibrotic area surrounding the granulomas. However, this may suggest functions for a2,81 other than binding collagen. Others have reported that a2,81 and a3,81 integrins may be involved in cell-cell adhesion. 55 Based on this information and our data together, a2 may mediate important cell-cell interactions between mononuclear cells involved in granuloma formation.

why the lung disease observed in patients with pulmonary sarcoidosis often resolves spontaneously. This discussion assumes the TGF-,81 detected is active. However, because the antibodies used to detect extracellular TGF -,81 are polyclonal, we suspect they detect both non-active and active forms. This is interesting particularly because ECMs may serve as a reservoir for several cytokines, including TGF -,81, and may even neutralize its activity.59 In summary, the similarities in distribution found for ECM, integrins, and TGF-,81 in sarcoid and experimental granulomas suggest that increased matrix expression and deposition, perhaps driven by local accumulation of TGF-,81,6o is a common mechanism for granuloma formation. We hypothesize that activation of mononuclear cells in the lung by unknown stimulants (in the case of sarcoidosis) or TD M (in the animal model) results in the accumulation of pro inflammatory cytokines that act in an autocrine and paracrine fashion to promote recruitment and activation of inflammatory cells. Increased matrix expression may modulate this response and perhaps participates in the localization of cells into the characteristic granuloma. Unopposed matrix deposition will favor remodeling of tissue with fibrosis. Although observations suggesting ECM-integrin and cytokine interactions can be made in human tissues, the TDM-induced granuloma model will allow us to test these interactions in vivo.

Transforming Growth Factor-,8i in Pulmonary Granulomatosis. Enhanced TGF -,81 expression was found in

The authors thank S. M. Aguayo, G. W. Staton Jr., and R. H. Ingram for useful discussions and comments on the manuscript and thank Trudy McDermott and Joanne Thompson-Crumble for their assistance in the preparation of tissue sections and immunohistochemical staining.

both clinical and experimental granulomatosis. Transforming growth factor-,81 is a polypeptide growth factor that up regulates matrix and matrix receptor gene expression in vitro. 16- 19 Transforming growth factor,81 has been implicated in pulmonary fibrosis where increased levels ofTGF -,81 protein and messenger RNA have been found. 4,56 Although TGF -,81 has been described within streptococcal cell wall-induced granulomas,57 little is known about its expression in other forms of pulmonary granulomatosis. Recently, Limper et a136 reported abundant TGF -,81 staining in Sarcoid granulomas. These prior observations and our current findings suggest that, in human sarcoidosis and TDM-induced pulmonary granulomatosis, TGF-,81 might be driving matrix gene expression, thereby promoting fibrosis. However, TGF,81 is considered an immunosuppressant modulator. For example, studies in knockout mice revealed that animals with a TGF-,81 loss-of-function gene mutation have remarkable infiltration of mononuclear leukocytes into multiple organs. 58 This and the observation that TGF -,81 is highly expressed early after administration of TDM in the animal model suggests that TGF-,81's main role is to promote a fibrotic reaction while diminishing inflammation. Control of the inflammatory response by TGF-,81 and other cytokines may explain THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

Acknowledgments

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