Clin. exp. Immunol. (1992) 89, 58-62
Circulating intercellular adhesion molecule-i (ICAM-1) antigen in sera of patients with idiopathic pulmonary fibrosis N. SHIJUBO, K. IMAI*, S. AOKI*, M. HIRASAWA, H. SUGAWARA, H. KOBA, M. TSUJISAKI*, T. SUGIYAMA*, Y. HINODA*, A. YACHI*, M. ASAKAWA & A. SUZUKI Department of Internal Medicine, Section 3 & *Section 1, Sapporo Medical College, Sapporo, Japan
(Acceptedfor publication 19 March 1992)
SUMMARY Intercellular adhesion molecule- I (ICAM- 1), a member of immunoglobulin supergene family with a five-domain structure, is known to play an important role in inflammatory diseases. An ELISA was developed using two MoAbs against human ICAM- t in order to detect the soluble shedding ICAM- l antigen in sera. We measured levels of circulating ICAM- l antigen in sera of patients with idiopathic pulmonary fibrosis (IPF), pulmonary sarcoidosis, hypersensitive pneumonitis, bacterial and mycoplasmal pneumonia, and inflammatory diseases of other organs. The results clearly demonstrated that IPF had significantly high levels of circulating ICAM-1 in sera as compared with other disorders or normal controls. Moreover, immunohistochemical analysis with MoAb against human ICAM-l disclosed that in IPF, the expression of ICAM-l was intensively enhanced on alveolar epithelial cells. These results suggest that ICAM-l may contribute to the pathogenesis of IPF.
Keywords ICAM-1 circulating ICAM-1 in sera idiopathic pulmonary fibrosis PATIENTS AND METHODS Monoclonal antibodies MoAb HA58 (IgGI) against ICAM-1 was developed in our laboratories [8] and MoAb CL207 (IgG1) against ICAM-l was developed as described elsewhere [9]. MoAbs were purified from ascitic fluid either by affinity chromatography on protein ASepharose [10], by ion exchange chromatography on DEAE or by caprylic acid precipitation [11]. MoAbs were biotinylated according to a published procedure [12]. These MoAbs were used for ELISA and immunohistochemistry.
INTRODUCTION Intercellular adhesion molecule-1 (ICAM-1), a member of immunoglobulin supergene family with a five-domain structure (an 80-110 kD glycoprotein) [1,2] has recently been characterized as one of the natural ligands to lymphocyte function associated antigen- 1 (LFA- 1) molecule, which is an alpha/beta heterodimer expressed on all leucocytes and a member of the integrin supergene family [1,3]. ICAM-l plays an important role in inflammatory diseases [4,5]. Release of cytokines, such as interferon (IFN), IL- 1, tumour necrosis factor (TNF), at sites of inflammation and immune response causes cell activation and results in augmented cellular expression of ICAM-1 [6]. Moreover, the expression of ICAM- 1 molecule on airway epithelial cells is reported to be up-regulated by IL- I f, TNF-ax or IFN-y [7] and ICAM-1 may contribute to the onset and progression of diseases characterized by chronic inflammation of the lower respiratory tract (i.e. idiopathic pulmonary fibrosis (IPF)). We recently established an ELISA using two MoAbs against ICAM-1, HA58 and CL207, to measure levels of circulating ICAM-1 antigen in sera [8]. We therefore measured levels of circulating ICAM- I in sera of patients with diffuse lung diseases including IPF and performed immunohistochemical analysis of ICAM-1 in tissue specimens from IPF patients. The results suggest that ICAM-I plays a crucial role in the pathogenesis of
Study population Idiopathic pulmonary fibrosis. Forty-five patients (aged 5078 years) with IPF were chosen in this study, and they had not received steroid therapy. Eight patients were histopathologically confirmed IPF from open lung biopsy specimens and nine patients from autopsy specimens. Other 28 patients were diagnosed on the following criteria [13-15]: (i) clinical criteria: no significant exposure to environmental agents, no symptoms to extrinsic allergic alveolitis, no history of chronic pulmonary infection or left ventricular failure, no evidence of collagen disease; (ii) radiologic criteria: a predominantly basal and subpleural reticulonodular pattern on chest radiogram and CT; (iii) physiologic criteria: a restrictive pattern of pulmonary function, decreased carbon monoxide diffusing capacity, hypoxymia at rest or on exercise. Pulmonary sarcoidosis. Seventeen untreated patients (aged 22-51 years) had a compatible clinical picture of sarcoidosis
IPF.
Correspondence: N. Shijubo, MD, Department of Internal Medicine (Section 3), Sapporo Medical College, S-i, W-16, Chuo-ku, Sapporo, 060, Japan.
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Circulating ICAM-J in IPF (compatible chest radiographic findings including enlargement of bilateral pulmonary hilar and/or paratracheal lymph nodes with lung parachymal infiltrates, and biopsy evidence of noncaseating epitheloid cell granuloma) without any evidence of mycobacterial, fungal or parasitic infection. None had a history of exposure to organic or inorganic materials known to cause granulomatous lung disorders. Hypersensitive pneomonitis (farmer's lung). Five patients (aged 35-52 years) were diagnosed hypersensitive pneumonitis with clinical information (symptoms such as fever, cough and dyspnoea appeared after exposure of antigens), compatible radiologic findings (reticulonodular shadows in bilateral lung fields), compatible laboratory data including elevated titres of precipitating antibodies to the offending antigen and compatible histopathological features from transbronchial lung biopsy. Pneumonia. Eighteen patients (aged 20-75 years) each had symptoms, i.e. fever, cough and a pneumonic shadow on chest radiogram. By sputum bacteriology and/or compatible findings of laboratory data 10 patients and eight patients were diagnosed bacterial and mycoplasmal pneumonia, respectively. They had no past history of respiratory diseases. Pancreatitis and cholecystitis. Fourteen patients were clinically confirmed pancreatitis and seventeen patients had cholecystitis. These patients had no history of respiratory disease and their chest radiograms showed no evidence of lung diseases. Healthy controls. Healthy controls (aged 35-78 years) were obtained from 48 volunteers among our departmental staff. They had no history of respiratory diseases and their chest radiograms and pulmonary function tests showed no evidence of respiratory diseases. Serum specimens and tissue specimens Serum specimens were collected from healthy controls and patients at our college hospital: IPF (n = 45), pulmonary sarcoidosis (n = 17), hypersensitive pneumonitis (n = 5), bacterial pneumonia (n=10), mycoplasmal pneumonia (n=8), inflammatory diseases of other organs (pancreatitis (n = 14) and cholecystitis (n = 17)), and cryopreserved at - 80°C until used. Three cryopreserved tissue specimens were obtained from open lung biopsy of patients with histopathological features of IPF. As normal control lung tissue, cryopreserved specimens were obtained by lobectomy from two patients with lung cancers. The lung tissue without tumour was confirmed histopathologically to be without interstitial thickening and inflammatory cell infiltration. These specimens were used in immuno-
histochemical analysis. ELISA for determination of soluble ICAM-I antigen ELISA using the FAST system (Becton Dickinson, Mountain View, CA) was employed to obtain a quantitative analysis of the antigen. This ELISA for measurement of soluble ICAM-l was developed in our laboratories [8]. Briefly, the beads, attached to the lid of a 96-well microtitre plate, were first incubated with purified MoAb CL207 (20 pg/ml) and then blocked with PBS, pH 7-4, containing 3% bovine serum albumin at 37°C for 120 min. Aliquots of serum samples diluted 1/200 in PBS were then incubated with the beads for 120 min. After being washed with PBS containing 0 05% Tween 20, the beads were incubated with biotinylated MoAb HA58 (10 pug/ml) for 120 mmn. Avidinconjugated peroxidase (Vector, Burlingame, CA) was dilutedl1/ 1000 in 0 05 M PBS with 0 5 M NaCl, pH 8 0, and incubated with
59
the beads for 60 min. The degree of substrate reaction was determined with OPD at 492 nm in a Micro-ELISA Autoreader MR580 (Dynatech, Cambridge, MA). Results were expressed as U/ml (I Unit corresponds to 2 ng of purified antigen which was obtained from the supernatant of cultured pancreatic carcinoma Panc- 1 cells) calculated from the titration curve of ICAM-I antigen. For the purification of the ICAM-l antigen, an affinity column was prepared by adding conjugated MoAb HA58 to cyanogen bromide-activated Sepharose-4B (Pharmacia, Uppsala, Sweden) at a concentration of 3 mg/ml. The gel filtration fractions containing ICAM-1 were collected from Sephacryl S-300 column (Pharmacia) and applied to the affinity column. The non-bound fractions were then eluted with 200 ml of PBS, pH 7-4, with 0 1 M NaHCO3 plus 0 5 M NaCl (pH 9 3). Subsequently bound antigen ICAM-1 was eluted with 01 M CH3COONa plus 0 5 M NaCl (pH 4-0), the resulting eluates being immediately neutralized with 2 M Tris-HCI, pH 8-0, and then dialysed against PBS. The purity of the antigen was monitored by SDS-PAGE (7 5%). All assays were performed in duplicate. The averages of circulating ICAM-1 antigen were expressed as the mean value + s.e.m. Mann-Whitney U-test was used to compare paired sets of data. The level of critical significance was assigned at P < 0-05. As a control MoAb for solid phase, MoAb MUSE 11 (IgGI) to human adenocarcinoma-associated antigen [16] was used. No reaction was obtained in this case.
Pathohistological and immunohistochemical analysis using MoAb against human ICAM-J antigen, HA58 Tissue specimens were obtained by operation (three IPF patients with open lung biopsy and two normal control lung tissues). Almost all specimens were fixed with 10% formalin and embedded in paraffin. After cutting, sections (5 pm) were stained with haematoxylin-eosin. Small thin sliced fresh specimens were frozen in liquid nitrogen. After cryo-sectioning, 5-pm sections were fixed with cold acetone for 10 min and either stained immediately or stored at - 30°C until used. The sections were incubated with MoAb, HA58 (undiluted culture supernatants) for 60 min at room temperature and washed three times with PBS for 30 min. The sections were then incubated with biotinylated goat anti-mouse serum. The biotinylated goat antimouse immunoglobulin was preapplied to human immunoglobulin-coupled Sepharose-4B to remove non-specific bindings to human tissues. After washing with PBS, the sections were reacted with avidin/biotin peroxidase complex (Vector). The enzyme reaction was developed as described previously [17]. Nuclei were lightly counterstained with haematoxylin. Isotypematched MoAb 1 C5 against human cervical adenocarcinoma of the uterus [17] was used as a negative control. RESULTS Measurement of circulating ICAM-1 antigen in the sera of patients with various diseases Regarding this ICAM-I ELISA, the inter-assay coefficient of variation calculated from five different assays during a period of 2 weeks was 7-2% and the mean intra-assay coefficient of variation obtained by testing the standard antigen on the same plate five times was found to be 4.6%. Using the ELISA, circulating ICAM-I antigen was measured in the sera of patients with IPF, pulmonary sarcoidosis,
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U/mt
-
.7 .0/10
_.
____
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sarcoidosis (Fig. 2). Three out of four IPF patients expired because of the disease progression (respiratory failure) and in the three deteriorated IPF patients the circulating ICAM-1 Sarwidosis levels were gradually elevated. The remaining IPF patient had 1/5 20% . continuously high levels of circulating ICAM-l. The successive analysis suggested the persistence of ICAM-1 shedding in the 0% sera from the IPF patients. In contrast, the patient with 0/8 0% sarcoidosis always exhibited low levels of circulating ICAM-1 antigen, and still had pulmonary infiltrative shadows up to the A _ _0/17 0% 2.14 14- -------------------last measurement of circulating ICAM-l antigen. 32/45 71%
aercoidositis pneumonitis pneumonia
the sera of four patients with IPF and one patient with
2/48
control IPF
Bacterial pneumonia Mycoplasmal
Next, we measured successively the circulating ICAM-1 in
M
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Healthy
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Cholecystitis _ _t *....
Pancreatitis
2/17
12%
2/14
14%
____
Fig. 1. Measurement of circulating ICAM-1 antigen. Serum samples patients with idiopathic pulmonary fibrosis (IPF), sarcoidosis, hypersensitive pneumonitis, bacterial pneumonia, mycoplasma pneumonia and inflammatory diseases of other organs (cut off value, 75 U/ml). were collected from
Death Death 4/Death En
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£/
v /Death 100
._____________________________ 3
9 6 12 Months after first admission Fig. 2. Successive analysis of circulating ICAM-1 antigen. Serum samples were obtained from four patients with idiopathic pulmonary fibrosis (IPF) (0) and one patient with sarcoidosis (v)
hypersensitive pneumonitis, pneumonia and inflammatory diseases of other organs as described in Patients and Methods. The results are shown in Fig. 1. The averages of circulating ICAM-l antigen in the sera of patients with IPF, pulmonary sarcoidosis,
hypersensitive pneumonitis, bacterial pneumonia, mycoplasmal pneumonia, pancreatitis, cholecystitis and healthy controls were 101 5 + 8-2 U/ml, 60-4 + 5.8 U/ml, 43-1 + 9.8 U/ml, 36-7 + 2 0 U/ ml, 34-7+2-9 U/ml, 49.8+4.1 U/ml, 50-2+4.1 U/ml and 50.9 + 1-8 U/ml, respectively. The circulating ICAM-l antigen levels alone in IPF were significantly high in comparison with those in healthy control (P < 0-001), but not in other disorders. The cut off value (mean value + 2 s.d.) was set as 75 U/ml, based on the data of 48 healthy control sera. In IPF, 71% of the circulating ICAM-l antigen levels exceeded the cut off value. In contrast, in pulmonary sarcoidosis, hypersensitive pneumonitis, bacterial pneumonia, mycoplasmal pneumonia, pancreatitis and cholecystitis, only 12%, 14%, 20%, 0%, 0% and 0% of the circulating ICAM-1 antigen levels were beyond the cut offvalue in the sera, respectively. Higher levels (more than 200 U/ml) of circulating ICAM-l antigen in sera were observed in four cases among only IPF patients as shown in Fig. 1.
Pathohistological and immunohistochemical analysis of the lung tissues in IPF with MoAb against ICAM-J, HA58 The pathohistological analysis of the three specimens obtained by open lung biopsy demonstrated usual interstitial pneumonia (UIP). UIP is known to be pathohistological features of IPF; interstitial thickening is patchy distributed with chronic inflammatory cell infiltration and micro-honeycombing and honeycombing are often present [13,18-20]. Since cryopreserved specimens were made to the exclusion of macroscopic honeycomb lesions, the cryopreserved biopsy specimens showed derangements of the interstitium and the epithelial cells; the interstitium was thickened by fibrosis and the normal epithelium had been replaced by cuboidal cells (histopathological features aof case shown in Fig. 3a). representative ................................ In order to clarify what type of cell expresses ICAM-1 antigen in the lung tissue of IPF, the immunohistochemical analysis with MoAb against ICAM-1, HA58, was performed using specimens of open lung biopsy from patients who were histopathologically confirmed UIP, as well as using normal control lung tissue specimens. As shown in Fig. 3b, in the lung tissues of IPF patients ICAM-1 molecule is intensively expressed on alveolar epithelial cells and ICAM-1 molecule is also expressed on vascular endothelial cells, while these cells failed to react with a negative control MoAb (Fig. 3c). In contrast, alveolar epithelial cells and vascular endothelial cells in normal lung tissues were not stained with MoAb against ICAM-1, HA58 (Fig. 3d).
DISCUSSION MoAb HA58 (IgG1) was established using IFN-y-treated colonic cancer BM314 cells as an immunogen as described previously [8]. This MoAb recognized ICAM- 1 molecule, which is a member of immunoglobulin supergene family, and one of the natural ligands to LFA-1 [3,21]. Moreover, MoAb HA58 appears to recognize the binding site (or close to it) of the ICAM- I molecule to LFA-1, since natural killer (NK) activity against K562 cells was strongly reduced by treatment of target cells with MoAb HA58, and the aggregation of ICAM-1 'and LFA-1 bearing cells was inhibited when the cells were treated with MoAb HA58 [8]. In contrast, CL207 was reported to recognize a distinct epitope which was not the binding site to LFA-1, since MoAb CL207 had no influence on NK activity or on aggregation of cells [9]. Because these two MoAbs recognize different epitopes of ICAM-l molecule, the ELISA was established in order to detect the circulating ICAM-l antigen in patients' sera [8].
Circulating ICAM-1 in IPF
61
0i
8,a
swan1 study
Fig. a o1(or
3. w
n
(m
c(
alveolar epithelial cells intensively expressed ICAM-1 molecule and on vascular endothelial cells ICAM-1 expression was observed
-W,44
(arrow heads). In contrast, alveolar epithelial cells and vascular endothelial cells in the normal lung tissue were not stained with MoAb against ICAM- 1.
IPF is known to be a chronic, fatal disorder affecting only the lower respiratory tract [13]. Although the term 'idiopathic' suggests that IPF may be a collection of fibrotic disorders of unknown aetiology, IPF is believed to be a specific disorder with characteristic features [18,19]. IPE usually begins in middle age presenting with dyspnoea with exercise, and has a 4-5 year course from onset to death. As the disease progresses, the lung becomes less and less able to transfer oxygen from air to blood in a normal fashion. The dyspnoea and problems with oxygen transfer result from derangements to the lung parenchyma that include injury to all of the cellular components of the alveolar wall and the accumulation of mesenchymal cells and their connective tissue products within alveolar walls and alveolar air-spaces [13,18,19]. The derangements of the lung parenchyma in IPF are caused by a chronic alveolitis which is dominated by alveolar macrophages and neutrophils but which also includes lymphocytes and eosinophils and, to a much lesser extent, basophils and mast cells [22,23]. Recent studies suggest that cytokines (i.e. TNF, IL-I1, etc.) are important mediators of pulmonary fibrosis. TNF mRNA in the total lung was reported to beincreased when lung injury was induced by bleomycin or silica [24,25]. Collagen deposition in silica-induced lung injury can be markedly decreased by administration of an anti-TNF antibody, indicating that the effects of
TNF are critical to the pathogenesis of these lesions. IL-i is released by macrophages as a result of exposure to inorganic dusts [26] and IL-1 increases prostaglandin synthesis by activating cyclo-oxygenase activity in mesenchymal cells [27]. These cytokines are known to enhance ICAM-1 expression on various cells as well as on vascular endothelial cells [6,21]. Wegner et al. [7] reported that the expression of ICAM- 1 on airway epithelial cells in an animal model was up-regulated by these cytokines in tvito and in vitro, and their results suggest that ICAM- 1 may contribute to the onset and progression of diseases characterized by chronic inflammation of the lower respiratory tract. Therefore, we examined levels of soluble ICAM- 1 antigen in sera of IPF using the ELISA, and immunohistochemically analysed open lung biopsy specimens of IPF patients employing MoAb against ICAM-l1 antigen, HA58. Although IPF is believed to be a specific disorder with characteristic features affecting only the lower respiratory tract, it is unknown whether IPF has an immunological pathway that is common to both the onset and progression of the disease. The present results clearly demonstrated that IPF had a significantly high level of circulating ICAM-1 in the sera, compared with pulmonary sarcoidosis and inflammatory diseases of other organs (Fig. 1). The successive analysis of circulating ICAM- 1 also suggested the persistence of ICAM- 1 shedding in the blood
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stream of the IPF patients. The immunohistochemical analysis showed that in IPF, the ICAM-1 expression is intensively enhanced on alveolar epithelial cells (Fig. 3b). Although speculative, the up-regulation of the ICAM-1 expression on these cells may be related to the elevation ofcirculating ICAM- I in the sera of IPF patients. In IPF, the derangements of the lung parenchyma are caused by a chronic alveolitis dominated by alveolar macrophages and neutrophils [22,23]. Barton et al. [4] reported that phorbol-esterinduced rabbit lung inflammation (interstitial thickening with intra-alveolar and interstitial inflammatory cell infiltration) was significantly reduced by pre-treatment with MoAb against ICAM- 1, suggesting that ICAM- 1 not only functions as a ligand for CD 18 but also can mediate, at least in part, the accumulation of neutrophils in inflammatory sites. It is unlikely that in IPF, ICAM- 1 on alveolar epithelial cells and/or vascular endothelial cells may mediate the migration of neutrophils to the inflammatory sites of the lungs. Whether or not ICAM- I may contribute to the onset and disease progression of IPF must be further elucidated.
ACKNOWLEDGMENTS This work was partly supported by Grant-in-Aid for Scientific Research K.I., M.T., A.Y.) from the Ministry of Education, Science and Culture, Japan and by Grant-in-Aid from the Ministry of Health and Welfare, Japan.
REFERENCES 1 Dustin ML, Staunton DL, Springer TA, Supergene families meet in the immune system. Immunol Today 1988; 9:213-5. 2 Makgoba MW, Sanders ME, Luce GEG et al. ICAM- l a ligand for LFA-1 dependent adhesion of B, T and myeloid cells. Nature 1988; 331:86-8. 3 Marlin SD, Springer TA. Purified intercellular adhesion molecule I (ICAM-1) is a ligand for lymphocyte function-associated antigen-I (LFA-1). Cell 1987; 51:813-9. 4 Barton RW, Rothlein R, Ksiazek J et al. The effect of antiintercellular adhesion molecule-I on phorbol-ester-induced rabbit lung inflammation. J Immunol 1989; 143:1278-82. 5 Albelda SM. Endothelial and epithelial cell adhesion molecules. Am J Respir Cell Mol Biol 1991; 4:195-203. 6 Rothlein R, Czajkowski M, O'Nell MM et al. Induction of intercellular adhesion molecule- 1 on primary and continuous cell lines by proinflammatory cytokines: regulation by pharmacologic agents and neutralizing antibodies. J Immunol 1988; 141:1665-9. 7 Wegner CD, Gundei RH, Reilly P et al. Intercellular adhesion molecule- I (ICAM- 1) in the pathogenesis of asthma. Science 1990; 247:456-9. 8 Tsujisaki M, Imai K, Hirata H et al. Detection of circulating intercellular adhesion molecule-I in malignant diseases. Clin Exp Immunol 1991; 81:3-8. 9 Maio M, Tessitoni G, Pinto A et al. Differential role of distinct determinants of intercellular adhesion molecule- 1 in immunologic phenomena. J Immunol 1989; 143:181-8.
10 Ey PL, Prowers SJ, Jenkins CR. Isolation of pure IgGI, IgG2a and IgG2b immunoglobulins from mouse serum using protein ASepharose. Immunochemistry 1978; 15:429-36. 11 Russo C, Callegaro L, Lanza E et al. Purification of IgG monoclonal antibody by caprylic acid precipitation. J Immunol Methods 1983; 65:269-71. 12 Guesdon JL, Ternynck T, Avrameas S. The use of avidinbiotin interaction in immunoenzymatic techniques. J Histochem Cytochem 1979; 79:1131-9. 13 Crystal RG, Fulmer JD, Roberts WC et al. Idiopathic pulmonary fibrosis: clinical, histologic, radiographic, scintigraphic, cytologic, and biochemical aspects. Ann Intern Med 1976; 85:769-88. 14 Fulmer JD, Roberts WC, Van Gal ER. Morphologic-physiologic correlates of the severity of fibrosis and degree of cellularity in idiopathic pulmonary fibrosis. J Clin Invest 1979; 63:665-76. 15 Muller NL, Miller RR, Webb WR et al. Fibrosing alveolitis: CTpathologic correlation. Radiology 1986; 160:585-8. 16 Ban T, Imai K, Yachi A. Immunohistologic and immunochemical characterization of a novel pancreatic cancer-associated antigen MUSEl l. Cancer Res 1989; 49:7141-6. 17 Koizumi M, Uede T, Shijubo N et al. New monoclonal antibody, LC5, reactive with human cervical adenocarcinoma of the uterus with immunodiagnostic potential. Cancer Res 1988; 48:6565-72. 18 Crystal RG, Gadek JE, Ferrans VJ et al. Interstitial lung disease: current concepts of pathogenesis, staging, and therapy. Am J Med 1981; 70:542-67. 19 Crystal RG, Bitterman PB, Rennard SI et al. Interstitial lung disease of unknown cause: disorders characterized by chronic inflammation of the lower respiratory tract. N Eng J Med 1984; 310:154-66, 23544. 20 Kuhn III C, Boldt J, Talmadge E et al. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am Rev Respir Dis 1989; 140:1693-703. 21 Dustin ML, Rothlein R, Bhan AK et al. Induction by IL-1 and interferon, tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol 1986; 137:24554. 22 Hunninghake GW, Gaedek JE, Kawanami 0 et al. Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage. Am J Pathol 1979; 97:149206. 23 Koegh BA, Crystal RG. Alveolitis: the key to the interstitial lung disorders. Thorax 1982; 37:1-10. 24 Piguet PF, Collart MA, Grau GE et al. Tumor necrosis factor/ cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis. J Exp Med 1989; 170:655-63. 25 Piguet PF, Collart MA, Grau GE et al. Requirement of tumor necrosis factor for development of silica-induced pulmonary fibrosis. Nature 1990; 344:2457. 26 Hartmann DP, Georgian MM, Oghiso Y et al. Enhanced interleukin activity following asbestos inhalation. Clin Exp Immunol 1984; 55:643-50. 27 Dayer JM, de Dochemonteix B, Burrus B et al. Human recombinant interleukin 1 stimulates collagenase and prostaglandin E2 production by human synovial cells. J Clin Invest 1986; 77:645-8.
Circulating intercellular adhesion molecule-i (ICAM-1) antigen in sera of patients with idiopathic pulmonary fibrosis N. SHIJUBO, K. IMAI*, S. AOKI*, M. HIRASAWA, H. SUGAWARA, H. KOBA, M. TSUJISAKI*, T. SUGIYAMA*, Y. HINODA*, A. YACHI*, M. ASAKAWA & A. SUZUKI Department of Internal Medicine, Section 3 & *Section 1, Sapporo Medical College, Sapporo, Japan
(Acceptedfor publication 19 March 1992)
SUMMARY Intercellular adhesion molecule- I (ICAM- 1), a member of immunoglobulin supergene family with a five-domain structure, is known to play an important role in inflammatory diseases. An ELISA was developed using two MoAbs against human ICAM- t in order to detect the soluble shedding ICAM- l antigen in sera. We measured levels of circulating ICAM- l antigen in sera of patients with idiopathic pulmonary fibrosis (IPF), pulmonary sarcoidosis, hypersensitive pneumonitis, bacterial and mycoplasmal pneumonia, and inflammatory diseases of other organs. The results clearly demonstrated that IPF had significantly high levels of circulating ICAM-1 in sera as compared with other disorders or normal controls. Moreover, immunohistochemical analysis with MoAb against human ICAM-l disclosed that in IPF, the expression of ICAM-l was intensively enhanced on alveolar epithelial cells. These results suggest that ICAM-l may contribute to the pathogenesis of IPF.
Keywords ICAM-1 circulating ICAM-1 in sera idiopathic pulmonary fibrosis PATIENTS AND METHODS Monoclonal antibodies MoAb HA58 (IgGI) against ICAM-1 was developed in our laboratories [8] and MoAb CL207 (IgG1) against ICAM-l was developed as described elsewhere [9]. MoAbs were purified from ascitic fluid either by affinity chromatography on protein ASepharose [10], by ion exchange chromatography on DEAE or by caprylic acid precipitation [11]. MoAbs were biotinylated according to a published procedure [12]. These MoAbs were used for ELISA and immunohistochemistry.
INTRODUCTION Intercellular adhesion molecule-1 (ICAM-1), a member of immunoglobulin supergene family with a five-domain structure (an 80-110 kD glycoprotein) [1,2] has recently been characterized as one of the natural ligands to lymphocyte function associated antigen- 1 (LFA- 1) molecule, which is an alpha/beta heterodimer expressed on all leucocytes and a member of the integrin supergene family [1,3]. ICAM-l plays an important role in inflammatory diseases [4,5]. Release of cytokines, such as interferon (IFN), IL- 1, tumour necrosis factor (TNF), at sites of inflammation and immune response causes cell activation and results in augmented cellular expression of ICAM-1 [6]. Moreover, the expression of ICAM- 1 molecule on airway epithelial cells is reported to be up-regulated by IL- I f, TNF-ax or IFN-y [7] and ICAM-1 may contribute to the onset and progression of diseases characterized by chronic inflammation of the lower respiratory tract (i.e. idiopathic pulmonary fibrosis (IPF)). We recently established an ELISA using two MoAbs against ICAM-1, HA58 and CL207, to measure levels of circulating ICAM-1 antigen in sera [8]. We therefore measured levels of circulating ICAM- I in sera of patients with diffuse lung diseases including IPF and performed immunohistochemical analysis of ICAM-1 in tissue specimens from IPF patients. The results suggest that ICAM-I plays a crucial role in the pathogenesis of
Study population Idiopathic pulmonary fibrosis. Forty-five patients (aged 5078 years) with IPF were chosen in this study, and they had not received steroid therapy. Eight patients were histopathologically confirmed IPF from open lung biopsy specimens and nine patients from autopsy specimens. Other 28 patients were diagnosed on the following criteria [13-15]: (i) clinical criteria: no significant exposure to environmental agents, no symptoms to extrinsic allergic alveolitis, no history of chronic pulmonary infection or left ventricular failure, no evidence of collagen disease; (ii) radiologic criteria: a predominantly basal and subpleural reticulonodular pattern on chest radiogram and CT; (iii) physiologic criteria: a restrictive pattern of pulmonary function, decreased carbon monoxide diffusing capacity, hypoxymia at rest or on exercise. Pulmonary sarcoidosis. Seventeen untreated patients (aged 22-51 years) had a compatible clinical picture of sarcoidosis
IPF.
Correspondence: N. Shijubo, MD, Department of Internal Medicine (Section 3), Sapporo Medical College, S-i, W-16, Chuo-ku, Sapporo, 060, Japan.
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Circulating ICAM-J in IPF (compatible chest radiographic findings including enlargement of bilateral pulmonary hilar and/or paratracheal lymph nodes with lung parachymal infiltrates, and biopsy evidence of noncaseating epitheloid cell granuloma) without any evidence of mycobacterial, fungal or parasitic infection. None had a history of exposure to organic or inorganic materials known to cause granulomatous lung disorders. Hypersensitive pneomonitis (farmer's lung). Five patients (aged 35-52 years) were diagnosed hypersensitive pneumonitis with clinical information (symptoms such as fever, cough and dyspnoea appeared after exposure of antigens), compatible radiologic findings (reticulonodular shadows in bilateral lung fields), compatible laboratory data including elevated titres of precipitating antibodies to the offending antigen and compatible histopathological features from transbronchial lung biopsy. Pneumonia. Eighteen patients (aged 20-75 years) each had symptoms, i.e. fever, cough and a pneumonic shadow on chest radiogram. By sputum bacteriology and/or compatible findings of laboratory data 10 patients and eight patients were diagnosed bacterial and mycoplasmal pneumonia, respectively. They had no past history of respiratory diseases. Pancreatitis and cholecystitis. Fourteen patients were clinically confirmed pancreatitis and seventeen patients had cholecystitis. These patients had no history of respiratory disease and their chest radiograms showed no evidence of lung diseases. Healthy controls. Healthy controls (aged 35-78 years) were obtained from 48 volunteers among our departmental staff. They had no history of respiratory diseases and their chest radiograms and pulmonary function tests showed no evidence of respiratory diseases. Serum specimens and tissue specimens Serum specimens were collected from healthy controls and patients at our college hospital: IPF (n = 45), pulmonary sarcoidosis (n = 17), hypersensitive pneumonitis (n = 5), bacterial pneumonia (n=10), mycoplasmal pneumonia (n=8), inflammatory diseases of other organs (pancreatitis (n = 14) and cholecystitis (n = 17)), and cryopreserved at - 80°C until used. Three cryopreserved tissue specimens were obtained from open lung biopsy of patients with histopathological features of IPF. As normal control lung tissue, cryopreserved specimens were obtained by lobectomy from two patients with lung cancers. The lung tissue without tumour was confirmed histopathologically to be without interstitial thickening and inflammatory cell infiltration. These specimens were used in immuno-
histochemical analysis. ELISA for determination of soluble ICAM-I antigen ELISA using the FAST system (Becton Dickinson, Mountain View, CA) was employed to obtain a quantitative analysis of the antigen. This ELISA for measurement of soluble ICAM-l was developed in our laboratories [8]. Briefly, the beads, attached to the lid of a 96-well microtitre plate, were first incubated with purified MoAb CL207 (20 pg/ml) and then blocked with PBS, pH 7-4, containing 3% bovine serum albumin at 37°C for 120 min. Aliquots of serum samples diluted 1/200 in PBS were then incubated with the beads for 120 min. After being washed with PBS containing 0 05% Tween 20, the beads were incubated with biotinylated MoAb HA58 (10 pug/ml) for 120 mmn. Avidinconjugated peroxidase (Vector, Burlingame, CA) was dilutedl1/ 1000 in 0 05 M PBS with 0 5 M NaCl, pH 8 0, and incubated with
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the beads for 60 min. The degree of substrate reaction was determined with OPD at 492 nm in a Micro-ELISA Autoreader MR580 (Dynatech, Cambridge, MA). Results were expressed as U/ml (I Unit corresponds to 2 ng of purified antigen which was obtained from the supernatant of cultured pancreatic carcinoma Panc- 1 cells) calculated from the titration curve of ICAM-I antigen. For the purification of the ICAM-l antigen, an affinity column was prepared by adding conjugated MoAb HA58 to cyanogen bromide-activated Sepharose-4B (Pharmacia, Uppsala, Sweden) at a concentration of 3 mg/ml. The gel filtration fractions containing ICAM-1 were collected from Sephacryl S-300 column (Pharmacia) and applied to the affinity column. The non-bound fractions were then eluted with 200 ml of PBS, pH 7-4, with 0 1 M NaHCO3 plus 0 5 M NaCl (pH 9 3). Subsequently bound antigen ICAM-1 was eluted with 01 M CH3COONa plus 0 5 M NaCl (pH 4-0), the resulting eluates being immediately neutralized with 2 M Tris-HCI, pH 8-0, and then dialysed against PBS. The purity of the antigen was monitored by SDS-PAGE (7 5%). All assays were performed in duplicate. The averages of circulating ICAM-1 antigen were expressed as the mean value + s.e.m. Mann-Whitney U-test was used to compare paired sets of data. The level of critical significance was assigned at P < 0-05. As a control MoAb for solid phase, MoAb MUSE 11 (IgGI) to human adenocarcinoma-associated antigen [16] was used. No reaction was obtained in this case.
Pathohistological and immunohistochemical analysis using MoAb against human ICAM-J antigen, HA58 Tissue specimens were obtained by operation (three IPF patients with open lung biopsy and two normal control lung tissues). Almost all specimens were fixed with 10% formalin and embedded in paraffin. After cutting, sections (5 pm) were stained with haematoxylin-eosin. Small thin sliced fresh specimens were frozen in liquid nitrogen. After cryo-sectioning, 5-pm sections were fixed with cold acetone for 10 min and either stained immediately or stored at - 30°C until used. The sections were incubated with MoAb, HA58 (undiluted culture supernatants) for 60 min at room temperature and washed three times with PBS for 30 min. The sections were then incubated with biotinylated goat anti-mouse serum. The biotinylated goat antimouse immunoglobulin was preapplied to human immunoglobulin-coupled Sepharose-4B to remove non-specific bindings to human tissues. After washing with PBS, the sections were reacted with avidin/biotin peroxidase complex (Vector). The enzyme reaction was developed as described previously [17]. Nuclei were lightly counterstained with haematoxylin. Isotypematched MoAb 1 C5 against human cervical adenocarcinoma of the uterus [17] was used as a negative control. RESULTS Measurement of circulating ICAM-1 antigen in the sera of patients with various diseases Regarding this ICAM-I ELISA, the inter-assay coefficient of variation calculated from five different assays during a period of 2 weeks was 7-2% and the mean intra-assay coefficient of variation obtained by testing the standard antigen on the same plate five times was found to be 4.6%. Using the ELISA, circulating ICAM-I antigen was measured in the sera of patients with IPF, pulmonary sarcoidosis,
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U/mt
-
.7 .0/10
_.
____
4
sarcoidosis (Fig. 2). Three out of four IPF patients expired because of the disease progression (respiratory failure) and in the three deteriorated IPF patients the circulating ICAM-1 Sarwidosis levels were gradually elevated. The remaining IPF patient had 1/5 20% . continuously high levels of circulating ICAM-l. The successive analysis suggested the persistence of ICAM-1 shedding in the 0% sera from the IPF patients. In contrast, the patient with 0/8 0% sarcoidosis always exhibited low levels of circulating ICAM-1 antigen, and still had pulmonary infiltrative shadows up to the A _ _0/17 0% 2.14 14- -------------------last measurement of circulating ICAM-l antigen. 32/45 71%
aercoidositis pneumonitis pneumonia
the sera of four patients with IPF and one patient with
2/48
control IPF
Bacterial pneumonia Mycoplasmal
Next, we measured successively the circulating ICAM-1 in
M
100
Healthy
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Cholecystitis _ _t *....
Pancreatitis
2/17
12%
2/14
14%
____
Fig. 1. Measurement of circulating ICAM-1 antigen. Serum samples patients with idiopathic pulmonary fibrosis (IPF), sarcoidosis, hypersensitive pneumonitis, bacterial pneumonia, mycoplasma pneumonia and inflammatory diseases of other organs (cut off value, 75 U/ml). were collected from
Death Death 4/Death En
200
£/
v /Death 100
._____________________________ 3
9 6 12 Months after first admission Fig. 2. Successive analysis of circulating ICAM-1 antigen. Serum samples were obtained from four patients with idiopathic pulmonary fibrosis (IPF) (0) and one patient with sarcoidosis (v)
hypersensitive pneumonitis, pneumonia and inflammatory diseases of other organs as described in Patients and Methods. The results are shown in Fig. 1. The averages of circulating ICAM-l antigen in the sera of patients with IPF, pulmonary sarcoidosis,
hypersensitive pneumonitis, bacterial pneumonia, mycoplasmal pneumonia, pancreatitis, cholecystitis and healthy controls were 101 5 + 8-2 U/ml, 60-4 + 5.8 U/ml, 43-1 + 9.8 U/ml, 36-7 + 2 0 U/ ml, 34-7+2-9 U/ml, 49.8+4.1 U/ml, 50-2+4.1 U/ml and 50.9 + 1-8 U/ml, respectively. The circulating ICAM-l antigen levels alone in IPF were significantly high in comparison with those in healthy control (P < 0-001), but not in other disorders. The cut off value (mean value + 2 s.d.) was set as 75 U/ml, based on the data of 48 healthy control sera. In IPF, 71% of the circulating ICAM-l antigen levels exceeded the cut off value. In contrast, in pulmonary sarcoidosis, hypersensitive pneumonitis, bacterial pneumonia, mycoplasmal pneumonia, pancreatitis and cholecystitis, only 12%, 14%, 20%, 0%, 0% and 0% of the circulating ICAM-1 antigen levels were beyond the cut offvalue in the sera, respectively. Higher levels (more than 200 U/ml) of circulating ICAM-l antigen in sera were observed in four cases among only IPF patients as shown in Fig. 1.
Pathohistological and immunohistochemical analysis of the lung tissues in IPF with MoAb against ICAM-J, HA58 The pathohistological analysis of the three specimens obtained by open lung biopsy demonstrated usual interstitial pneumonia (UIP). UIP is known to be pathohistological features of IPF; interstitial thickening is patchy distributed with chronic inflammatory cell infiltration and micro-honeycombing and honeycombing are often present [13,18-20]. Since cryopreserved specimens were made to the exclusion of macroscopic honeycomb lesions, the cryopreserved biopsy specimens showed derangements of the interstitium and the epithelial cells; the interstitium was thickened by fibrosis and the normal epithelium had been replaced by cuboidal cells (histopathological features aof case shown in Fig. 3a). representative ................................ In order to clarify what type of cell expresses ICAM-1 antigen in the lung tissue of IPF, the immunohistochemical analysis with MoAb against ICAM-1, HA58, was performed using specimens of open lung biopsy from patients who were histopathologically confirmed UIP, as well as using normal control lung tissue specimens. As shown in Fig. 3b, in the lung tissues of IPF patients ICAM-1 molecule is intensively expressed on alveolar epithelial cells and ICAM-1 molecule is also expressed on vascular endothelial cells, while these cells failed to react with a negative control MoAb (Fig. 3c). In contrast, alveolar epithelial cells and vascular endothelial cells in normal lung tissues were not stained with MoAb against ICAM-1, HA58 (Fig. 3d).
DISCUSSION MoAb HA58 (IgG1) was established using IFN-y-treated colonic cancer BM314 cells as an immunogen as described previously [8]. This MoAb recognized ICAM- 1 molecule, which is a member of immunoglobulin supergene family, and one of the natural ligands to LFA-1 [3,21]. Moreover, MoAb HA58 appears to recognize the binding site (or close to it) of the ICAM- I molecule to LFA-1, since natural killer (NK) activity against K562 cells was strongly reduced by treatment of target cells with MoAb HA58, and the aggregation of ICAM-1 'and LFA-1 bearing cells was inhibited when the cells were treated with MoAb HA58 [8]. In contrast, CL207 was reported to recognize a distinct epitope which was not the binding site to LFA-1, since MoAb CL207 had no influence on NK activity or on aggregation of cells [9]. Because these two MoAbs recognize different epitopes of ICAM-l molecule, the ELISA was established in order to detect the circulating ICAM-l antigen in patients' sera [8].
Circulating ICAM-1 in IPF
61
0i
8,a
swan1 study
Fig. a o1(or
3. w
n
(m
c(
alveolar epithelial cells intensively expressed ICAM-1 molecule and on vascular endothelial cells ICAM-1 expression was observed
-W,44
(arrow heads). In contrast, alveolar epithelial cells and vascular endothelial cells in the normal lung tissue were not stained with MoAb against ICAM- 1.
IPF is known to be a chronic, fatal disorder affecting only the lower respiratory tract [13]. Although the term 'idiopathic' suggests that IPF may be a collection of fibrotic disorders of unknown aetiology, IPF is believed to be a specific disorder with characteristic features [18,19]. IPE usually begins in middle age presenting with dyspnoea with exercise, and has a 4-5 year course from onset to death. As the disease progresses, the lung becomes less and less able to transfer oxygen from air to blood in a normal fashion. The dyspnoea and problems with oxygen transfer result from derangements to the lung parenchyma that include injury to all of the cellular components of the alveolar wall and the accumulation of mesenchymal cells and their connective tissue products within alveolar walls and alveolar air-spaces [13,18,19]. The derangements of the lung parenchyma in IPF are caused by a chronic alveolitis which is dominated by alveolar macrophages and neutrophils but which also includes lymphocytes and eosinophils and, to a much lesser extent, basophils and mast cells [22,23]. Recent studies suggest that cytokines (i.e. TNF, IL-I1, etc.) are important mediators of pulmonary fibrosis. TNF mRNA in the total lung was reported to beincreased when lung injury was induced by bleomycin or silica [24,25]. Collagen deposition in silica-induced lung injury can be markedly decreased by administration of an anti-TNF antibody, indicating that the effects of
TNF are critical to the pathogenesis of these lesions. IL-i is released by macrophages as a result of exposure to inorganic dusts [26] and IL-1 increases prostaglandin synthesis by activating cyclo-oxygenase activity in mesenchymal cells [27]. These cytokines are known to enhance ICAM-1 expression on various cells as well as on vascular endothelial cells [6,21]. Wegner et al. [7] reported that the expression of ICAM- 1 on airway epithelial cells in an animal model was up-regulated by these cytokines in tvito and in vitro, and their results suggest that ICAM- 1 may contribute to the onset and progression of diseases characterized by chronic inflammation of the lower respiratory tract. Therefore, we examined levels of soluble ICAM- 1 antigen in sera of IPF using the ELISA, and immunohistochemically analysed open lung biopsy specimens of IPF patients employing MoAb against ICAM-l1 antigen, HA58. Although IPF is believed to be a specific disorder with characteristic features affecting only the lower respiratory tract, it is unknown whether IPF has an immunological pathway that is common to both the onset and progression of the disease. The present results clearly demonstrated that IPF had a significantly high level of circulating ICAM-1 in the sera, compared with pulmonary sarcoidosis and inflammatory diseases of other organs (Fig. 1). The successive analysis of circulating ICAM- 1 also suggested the persistence of ICAM- 1 shedding in the blood
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stream of the IPF patients. The immunohistochemical analysis showed that in IPF, the ICAM-1 expression is intensively enhanced on alveolar epithelial cells (Fig. 3b). Although speculative, the up-regulation of the ICAM-1 expression on these cells may be related to the elevation ofcirculating ICAM- I in the sera of IPF patients. In IPF, the derangements of the lung parenchyma are caused by a chronic alveolitis dominated by alveolar macrophages and neutrophils [22,23]. Barton et al. [4] reported that phorbol-esterinduced rabbit lung inflammation (interstitial thickening with intra-alveolar and interstitial inflammatory cell infiltration) was significantly reduced by pre-treatment with MoAb against ICAM- 1, suggesting that ICAM- 1 not only functions as a ligand for CD 18 but also can mediate, at least in part, the accumulation of neutrophils in inflammatory sites. It is unlikely that in IPF, ICAM- 1 on alveolar epithelial cells and/or vascular endothelial cells may mediate the migration of neutrophils to the inflammatory sites of the lungs. Whether or not ICAM- I may contribute to the onset and disease progression of IPF must be further elucidated.
ACKNOWLEDGMENTS This work was partly supported by Grant-in-Aid for Scientific Research K.I., M.T., A.Y.) from the Ministry of Education, Science and Culture, Japan and by Grant-in-Aid from the Ministry of Health and Welfare, Japan.
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