Mast Cell-Associated Basic Fibroblast Growth Factor in the Fibrotic Response to Environmental Toxins

Arch Allergy Appl Immunol1982; 67:175-80 20 Newman LS, Mroz MM, Schumacher B, eta!. Beryllium sensitization precedes chronic beryllium disease. Am Rev Respir Dis 1992; 145(suppl):A324 21 Waksman BH. Discussion of "the diagnosis of beryllium disease with special reference to the patch test" by GH Curtis. Arch Ind Health 1959; 19:154-56 22 Jones-Williams W, Williams WR. Value of beryllium lymphocyte transformation tests in chronic beryllium disease and in potentially exposed workers. Thorax 1983; 38:41-4 23 Kreiss K, Miller F , Newman LS, eta!. Chronic beryllium disease: from the workplace to cellular immunology, molecular immunogenetics, and back. Clin Immunol Immunopathol1994; 71:123-29 24 Price CD, Pugh A, Pioli EM, et a!. Beryllium macrophage migration inhibition test. Ann NY Acad Sci 1976; 278:204-11 25 Epstein PE, Dauber JH, Rossman MD, et a!. Bronchoalveolar lavage in a patient with chronic berylliosis: evidence for hypersensitivity pneumonitis. Ann Intern Med 1982; 97:213-16 26 Rossman MD, Kern JA, Elias JA, eta!. Proliferative response of bronchoalveolar lymphocytes to beryllium: a test for chronic beryllium disease. Ann Intern Med 1988; 108:687-93 27 Newman LS, Kreiss K, King TE Jr, eta!. Pathologic and immunologic alterations in early stages of beryllium disease: re-examination of disease definition and natural history. Am Rev Respir Dis 1989; 139:1479-86 28 Mroz MM, Kreiss K, Lewtte DC, et a!. Reexamination of the blood lymphocyte transfonnation test in the diagnosis of chronic beryllium disease. J Allergy Clin Immunol 1991; 88:54-60 29 Bargon J, Kronenberger H , Bergmann L, et a!. Lymphocyte transformation test in a group of foundry workers exposed to beryllium and non-exposed controls. Eur J Respir Dis 1986; 69(suppl 136):211-15 30 Kreiss K, Newman LS, Mroz MM, et a!. Screening blood test identifies subclinical beryllium disease. J Occup Med 1989; 31:603-08 31 Kreiss K, Mroz MM, Zhen B, et a!. Epidemiology of beryllium sensitization and disease in nuclear workers. Am H.ev Respir Dis 1993; 148:985-91 32 Kreiss K, Wasserman S, Mroz MM, et a!. Beryllium disease screening in the ceramics industry: blood test p erfonnance and exposure-disease relations. J Occup Med 1993; 35:267-74 33 Newman LS, lloyd J, DaniloiTE. The natural history ofberyllium sensitization and chronic beryllium disease, 1994. Environ Health Perspect (in press) 34 Newman LS. To Be2+ or not to Be 2 +: relating immunogenetics to occupational exposure. Science 1993; 262:197-98 35 Saltini C, Winestock K, Kirby M, et a!. Maintenance of alveolitis in patients with chronic beryllium disease by beryllium-specific helper T cells. N Eng! J M ed 1989; 320:1103-09 36 Bossman MD, Yan H-C, Murray RK, et a!. Chronic beryllium disease: an immune response by restricted subfamilies ofT cells. Am H.ev Respir Dis 1992; 145:A415 37 Comment CE, Kotzin BL, Schumacher BA, eta!. Preferential use ofT cell antigen receptors in beryllium disease. Am J R espir Crit Care Med 1994; 149:A264 38 Romagnoli P, Spinas GA, Sinigaglia F. Gold-specific T cells in rheumatoid arthritis patients treated with gold. J Clin Invest 1992; 89:254-58 39 Sinigaglia F , Scheidegger D , Garotta G, et a!. Isolation and characterization of Ni-specific T cell clones from patients with Ni-contact dermatitis. J Immunol 1985; 135:3929-32 40 Emtestam L, Marcusson JA, Moller E. HLA class II restriction specificity for nickel-reactive T lymphocytes. Acta Dermatol Venereal Stockh 1988; 68:395-401 41 Kauppinen T , Partanen T. Use of plant-and-period specific jobexposure matrices in studies on occupational cancer. Scand J Work Environ Health 1988; 14:161-67

42 Bost TW, H.iches DWH, Schumacher B, et a!. Alveolar macrophages from patients with beryllium disease and sarcoidosis express increased levels of mRNA for TNF -ct and IL-6 but not IL1(3. Am J H.espir Cell Mol Bioi 1994; 10:506-13 43 Tinkle SS, Schwitters PW, Newman LS. Beryllium stimulates release of TNF-ct, IL-6, IL-2 and IFN--y but not IL-4 from bronchoalveolar lavage cells in chronic beryllium disease. Environ Health Perspect (in press ) 44 Barna BP, Deodhar SD, Chiang T, eta! . Experimental berylliuminduced lung disease: I. Differences in immunologic response to beryllium compounds in strains 2 and 13 guinea pigs. Int Arch Allergy Appl Immunol 1984; 73:42-8 45 Barna BP, Deodhar SD, Gautam S, et a!. Experimental beryllium-induced lung disease: II. Analyses of bronchial lavage cells in strains 2 and 13 guinea pigs. Int Arch Allergy Appl Immunol1984; 73:49-55 46 Huang H , M eyer KC , Kubai L, et al. An immune model of beryllium-induced pulmonary granulomata in mice: histopathology, immune reactivity, and flow-cytometric analysis of bronchoalveolar lavage-derived cells. Lab Invest 1992; 67:138-46 47 McConnochie K, Williams WR, Kilpatrick GS, et a!. Chronic beryllium disease in identical twins. Br J Dis Chest 1988; 82:431-35 48 Richeldi L, Sorrentino R, Saltini C. HLA-DPJ31 glutamate 69: a genetic marker of beryllium disease. Science 1993; 262:242-44

Mast Cell-Associated Basic Fibroblast Growth Factor in the Fibrotic Response to Environmental Toxins* The Beryllium Model Yoshikazu Inoue, MD, PhD; Mark Comebise, MS; Elaine Daniloff; Jenifer Lloyd, DVM, MSPH; Sally Tinkle, PhD; Talmadge E. King Jr., MD, FCCP; Lee S. Newman, MD, FCCP

P

ulmonary interstitial fibrosis is a devastating outcome of the chronic inflammatory response to inhaled toxins, including metals such as beryllium . Basic fibroblast growth factor (b FGF) is a potent stimulator of fibroblast, endothelial cell, and smooth muscle cell proliferation and has been linked with fibroproductive and angiogenic responses. We hypothesized that excess production of bFGF occurs in chronic beryllium disease (CBD) as well as in "idiopathic" lung disorders . Furthermore, we hypothesized that mast cells may be the source of key profibrotic growth factors in the lungs in such diseases. To test these hypotheses, we performed immunohistochemical staining and computer-assisted morphometric analysis of lung tissue from CBD (n=l3), sarcoidosis (Sar; n=lO), idiopathic pulmonary fibrosis (IPF; n=ll), normal control subjects (Nor; n=ll), and nondiseased patients with beryllium sensitization (BeS; n=8) . We observed immunoreactive bFGF in tryptase+ mast cells in both normal and diseased lung. In addition to mast cells, bFGF localized to basement membrane and endothelial cells, but not to CD68+ macrophages. Quantitation of the volume density of (Vv) bFGF+ cells and Vv tryptase+ cells demonstrated a statisti*From the Occupational/Environmental Medicine Division, National J ewish Center for Immunology & Respiratory Medicine, University of Colorado Health Sciences Center, Denver. CHEST I 109 I 3 I MARCH, 1996 I Supplement

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cally significant increase in these cell populations in the lungs of patients with CBD, Sar, and IPF compared to Nor and BeS lung tissue. The highest values were observed in IPF, consistent with the severe extent of fibrotic reaction in that disorder. There was a significant correlation between Vv bFGF+ cells and Vv tryptase+ cells (r=0.87; p<0.001). Basic FGF+ mast cells colocalized to the periphery of granulomas (r=0.53; p
Basic Fibroblast Growth Factor in Fibrosing Alveolitis Induced by Oxygen Stress* Philip L. Sannes, PhD; Jody Khosla, MS; Sherwood Johnson, DVM; Malgorwta Goralska, PhD; Chris McGahan, PhD; and Moncia Menard, DVM

of exposure of lungs to certain environA characteristic mental pollutants is irreversible fibrotic change, which

results, at least in part, from the ineffective replacement of damaged epithelium within the pulmonary alveolus. The surfactant-producing type II cell is known to play a central role in this process by virture of its capacity to divide and renew itself as well as differentiate into type I cells. There are a number of key determinants that influence type II cell behavior, including components of extracellular matrices (ECMs) and a select group of growth factors. Basic fibroblast growth factor (bFGF) has been shown to stimulate the uptake of DNA precursors, and therefore has been suggested to be an effector of type II cell proliferation. 1•2 bFGF is known to be present in the alveolar basement membrane (AB M), 3 presumably bound to heparan sulfate proteolgycan, where it could be liberated by enzymatic activity following damage and affect local proliferative events4 Modulation of these events likely involves up-regulation and down-regulation of biosynthesis of key growth factors by relevant cells responding to inflammatory mediators. We hypothesized that bFGF is such a key growth factor, and that it is biosynthesized by type II cells in response to bFGF and transforming growth factor (3 (TGF(3). The goals of this study were twofold: (l) to determine whether the expression of bFGF product in the lung changed during the early progression of fibrosing alveolitis, and (2) to determine whether isolated type II cells biosynthesized bFGF. To address the first, we employed an appropriate disease model using rats exposed to continuous 85% oxygen for 14 days to evaluate changes in distribution of bFGF in whole lung

*From the De[Jartment of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State U nivers1t):, Raleig_h. Supported by Public Health Service grant HL-44497 and EY04900. Reprint requests: Philip Sannes, PhD, Anatomy, Physiology and Radiology, North Carolina State University Veterinary College, 4700 1-Hllsborough Street, Raleigh, NC 27606

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sections by immunohistochemistry. To attempt to define the cellular source of this potentially important effector molecule, type II cells were isolated from normal rats, cultured under different conditions, and expression of bFGF was examined using Northern analysis and immunohistochemistry. Regulation ofbFGF in type II cells was also examined in a preliminary way by culturing cells on different matrix substrata in the presence of selected growth factors. MATERIALS AND METHODS

Sprague-Dawley rats were exposed to either continuous humidified air or 85% oxygen for 14 days. Animals were killed at days 3, 7, 14, or 28 days and the lungs were insufflated with 4% buffered paraformaldehyde for 2 h. Specimens were prepared for routine paraffin embedment, sectioned, and immunostained using a monoclonal antibody to bovine bFGF (UBI; Lake Placid, NY). For molecular studies, riboprobes were generated with a bFGF complementary DNA from rat brain (870 bp; obtained from T. Kurokawa; Takeda Chemical Industries Ltd; Osaka, JAPAN) cloned into the EcoRI site of PBR322 and subcloned into EcoR1 site of pBluescript II sk (Stratagene). This plasmid contained promoters for in vitro transcription of the 32 P riboprobes. The direction of the subcloned fragment was confirmed by sequencing. For preparation of the riboprobe, the plasmid was linearized with Xba I for the sense probe and Hindlll for antisense. An 185 probe was obtained (from ATCC) and linearized with EcoRV for preparation of riboprobes. All riboprobes were made using a kit (Riboprobe Gemini; Promega) according to the instructions of the manufacturer. They were then used to probe Northern blots of RNA purified from isolated rat type II cells cultured under different conditions. Cells were plated on type I collagen gel that had been dried and coated with 24 pgtcm 2 of either sulfated or chemically desulfated high molecular weight heparin. They were then exposed to either 100 nglmL bFGF or 1 nglmL TGFf3 in serum-free hormonally defined (SF) media or SF media alone as indicated. The bFGF probe hybridized with the predicted 4.3 kb RNA, and was a single species, much like that seen in adult whole brain and lung. Cultured type II cells processed in parallel with RNA studies were prepared for whole cell immunohistochemistry. RESULTS

lmmunostaining of oxygen-exposed rat lungs demonstrated that bFGF appears to be liberated from storage sites between days 3 to 7 of exposure as evidenced by obvious discontinuities in the ABM , and between days 7 and 14, as significant increase in immunoreactivity for bFGF within the ABM. Short-term culture indicated that type II cells biosynthesize bFGF, as demonstrated by its messenger RNA detected by Northern analysis and product immunodetected in whole cells. Cells cultured 72 to 120 h expressed significantly higher levels of bFGF messenger RNA than those freshly isolated. Cells exposed to bFGF had reduced levels of message when cultured on heparin substrata, but a lesser reduction on desulfated heparin, compared with collagen I alone. TGF(3 exposed cells had reduced message when cultured on desulfated heparin, but a lesser reduction on native heparin compared with collagen I. Immunostaining for bFGF product in cells prepared in parallel supported these observations for the most part, despite their strictly qualitative nature. DISCUSSION

This study demonstrated that bFGF appears altered in the Thomas L. Petty 38th Annual Aspen Lung Conference