Kazuhiro Iyonaga

Monocyte chemoattractant protein-1 in idiopathic pulmonary fibrosis and other interstitial lung diseases.

Macrophages play a crucial role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). To examine the mechanisms for increased monocyte/macrophage recruitment in IPF and nonIPF interstitial lung diseases (nonIPF) the localization of monocyte chemoattractant protein-1 (MCP-1) was investigated in 14 cases of IPF, seven cases of nonIPF, and seven normal control lungs (CTRL) by immunohistochemistry using a specific anti-MCP-1 monoclonal antibody, F9. By double immunohistochemical staining using F9 and one of the cell type specific antibodies significant differences in the staining pattern of MCP-1 were observed between IPF and nonIPF. In IPF MCP-1 was observed in cuboidal and flattened metaplastic epithelial cells, alveolar macrophages, and vascular endothelial cells. In contrast, no epithelial cells were stained for MCP-1 in nonIPF cases, although alveolar macrophages and vascular endothelial cells were labeled. Northern hybridization analysis of selected cases showed marked expression of MCP-1 messenger RNA (mRNA) in IPF and nonIPF compared with CTRL. These findings suggest that the MCP-1 production in IPF and nonIPF plays an important role in the recruitment of monocyte/macrophages. Monocyte chemoattractant protein-1 production by epithelial cells in IPF may be caused by the metaplastic nature of the epithelial cells and may be one of the key factors inducing the irreversible progression of IPF.

Differentiation, maturation, and survival of dendritic cells by osteopontin regulation

ABSTRACT Dendritic cells (DCs) are antigen-presenting cells with the ability to induce primary immune responses necessary in innate immunity and adaptive immunity. Osteopontin (OPN) is a secreted acidic phosphoprotein containing an arginine-glycine-aspartate sequence and has been suggested to play an important role in early cellular immune responses. The interaction between DCs and OPN has not been clarified. We hypothesized that there is an important interaction between DCs and OPN, which is an indispensable extracellular matrix component in early cellular immune responses. Human monocyte-derived DCs synthesized OPN especially during the differentiation from monocytes to immature DCs. By blocking of OPN with anti-OPN antibody, cultured DCs became smaller and expressed lower levels of costimulatory molecules and major histocompatibility complex class II antigens than untreated DCs. Furthermore, DCs treated with anti-OPN antibody easily underwent apoptosis. These results suggest that human DCs can produce OPN and that OPN may play a role in the differentiation, maturation, and survival of DCs by autocrine and/or paracrine pathways.

Expression of matrix metalloproteinases in pigs with hyperoxia-induced acute lung injury

The aim of this study was to determine the role of matrix metalloproteinases (MMPs) in the pathogenesis of acute lung injury induced by hyperoxia. Twenty-three pigs were exposed in sealed cages to >80% oxygen (for 24-120 h) or room air. Correlation between MMP-2/MMP-9 activity, measured by gelatin zymography in bronchoalveolar lavage fluid (BALF), and the histological findings and pathological parameters were examined in detail. Sources of these MMPs in the hyperoxic lung were analysed by immunohistochemistry. The histological progression of acute lung injury in this model ranged from the early exudative to the early proliferative phase of diffuse alveolar damage (DAD). MMP-2 and -9 activities were elevated under prolonged hyperoxic exposure. MMP-9 activity correlated significantly with the oxygen tension in arterial blood/inspiratory oxygen fraction, the lung wet-to-dry weight ratio, and the number of neutrophils in BALF, whereas MMP-2 activity did not correlate at all with these factors. MMP-9 activity correlated more closely with the pathological findings of DAD than did MMP-2 activity. Strong MMP-9 expression was observed in neutrophils, alveolar macrophages as well as alveolar lining epithelial cells. These results suggest that matrix metalloproteinase. which may derive from neutrophils recruited into airspaces, plays an important role in the pathogenesis of hyperoxic diffuse alveolar damage

Alterations in cytokeratin expression by the alveolar lining epithelial cells in lung tissues from patients with idiopathic pulmonary fibrosis.

It is generally recognized that epithelial cytokeratins (CKs) are expressed in tissue‐specific patterns and reflect differentiation, functional specialization, and pathological alterations of the cells. Differential epithelial cell types can thus be distinguished from each other by their selective expression of particular sets of CKs. To determine the characteristics of metaplastic and hyperplastic changes of alveolar‐lining epithelial cells in the lungs of idiopathic pulmonary fibrosis (IPF), the expression of individual CKs was studied immunohistochemically using monospecific anti‐CK monoclonal antibodies (anti‐CKs 7, 8, 10, 13, 14, 16, 17, 18, 19). Biopsy specimens from 17 patients with IPF and normal lung tissues (NL) from seven patients with lung cancer were studied. In the IPF specimens, several kinds of altered epithelial cells were observed, which showed characteristic changes in CK expression compared with NL, especially CKs 8, 14, and 17. Hyperplastic type II cells expressed increased CKs 7, 8, and 19, but not CK 17; flattened or stratified squamous metaplastic cells expressed increased CKs 17 and 14, co‐expressed with CKs 7, 8, and 19; bronchiolar metaplastic cells expressed increased CKs 7, 8, and 19, co‐expressed with CKs 14 and 17; cuboidal metaplastic cells expressed increased CKs 7, 8, 17, and 19. The quantification of individual CKs in the tissues by enzyme‐linked immunosorbent assay revealed increased expression of CKs 8, 14, and 17 in IPF lung tissues compared with NL. These results were consistent with the immunohistochemical observations. The hyperplastic and bronchiolar metaplastic phenotypes were characterized by their increased expression of simple CKs without CK alteration. The squamous metaplastic phenotype showed CK alterations, with the appearance of CKs 17 and 14. Epithelial cells are thus altered not only in shape, but possibly also in differentiation and function, with potential implications for the pathogenesis of IPF.

Use of tacrolimus, a potent antifibrotic agent, in bleomycin-induced lung fibrosis

Idiopathic pulmonary fibrosis has a poor prognosis and few efficacious treatments. The immunosuppressant cyclosporin A has been shown to inhibit tumour growth factor (TGF)-β-induced collagen deposition in vitro, and is widely used in Japan as a potent antifibrotic agent. Tacrolimus (FK506) is another attractive immunosuppressant, which may be useful in the treatment of pulmonary fibrosis. The aim of the present study was to elucidate the antifibrotic effect of FK506. The inhibitory effect of FK506 on collagen synthesis in cultured lung fibroblastic cells, TIG-3-20, and its antifibrotic effect on bleomycin (BLM)-induced pulmonary fibrosis in mice was investigated. FK506 inhibited TGF-β-induced collagen synthesis, and suppressed the expression of TGF-β type I receptor (TβR-I) in TIG-3-20 cells. Consistent with the in vitro findings, FK506 treatment starting on day 6 attenuated BLM-induced pulmonary fibrosis, in part, via reduced TβR-I expression. FK506 treatment in the acute BLM injury phase unexpectedly increased pro-inflammatory cytokine levels in bronchoalveolar lavage fluid and enhanced lung injury, resulting in poor survival. In conclusion, the present results suggest that FK506 has a potent antifibrotic effect and may be useful for the treatment of pulmonary fibrosis, although its use in the acute inflammatory phase may exacerbate lung injury.

Functional roles of MCP-1 in Propionibacterium acnes-induced, T cell-mediated pulmonary granulomatosis in rabbits.

The immunological manifestation of granuloma formations in humans largely depends on the delayed‐type hypersensitivity response. We investigated the involvement of monocyte chemoattractant protein‐1 (MCP‐1) in a rabbit model of T cell‐mediated pulmonary granulomatosis. Intravenous injection of Propionibacterium acnes (P. acnes) into sensitized rabbits induced massive and diffuse pulmonary granulomas. Levels of MCP‐1 in sera and bronchoalveolar lavage fluids (BALF) peaked before the granuloma formation reached the peak (on days 1 and 3 after challenge, respectively). Chemotactic activities toward monocytes and T cells in BALF were inhibited by anti‐MCP‐1 IgG by 80 and 36%, respectively. The phenotypic analysis of the migrating T cells revealed that activated and memory T cells rather than naive cells were preferentially attracted to the BALF. Administration of anti‐MCP‐1 antiserum inhibited the development of granuloma formation in both size and number, the numbers of infiltrating leukocytes in BALF, the expression of adhesion molecules on peripheral monocytes/T cells, and on macrophages/T cells in BALF, and the production of TNF‐α in the lung. Anti‐MCP‐1 resulted in a trend toward decreased level of IL‐1β in the lung. The inhibition of the production of these cytokines appeared to be induced indirectly through the inhibition of the recruitment of macrophages that produce these cytokines. The results suggest important roles of MCP‐1 in the development of granuloma formation in this model through the attraction and activation of specific types of cells. J. Leukoc. Biol. 65:482–491; 1999.

A novel monoclonal antibody, RM-4, specifically recognizes rat macrophages and dendritic cells in formalin-fixed, paraffin-embedded tissues

SummaryAn anti-rat macrophage/dendritic cell monoclonal antibody, RM-4, was produced using a homogenate of silica-induced lung granulomas of rat as immunogen. Immunohistochemistry demonstrated that RM-4 was specific for macrophage and dendritic cell populations residing in various organs and tissues. It did not react with any cells other than macrophage/dendritic cells. In the double staining of the spleen, RM-4-positive macrophages showed wider distribution than those of the four other anti-rat macrophage monoclonal antibodies compared. The immunoreactivity of RM-4 was well preserved not only in frozen sections but also in formalin-fixed, paraffin-embedded tissues. The isotype of the monoclonal antibody was IgG1 kappa and its antigen molecular weight was 46 kDa. Immunoelectron microscopy revealed positive reaction products for RM-4 on the membrane of endosomes and lysosomes in macrophages and epidermal Langerhans cells. Reaction intensity increased after thioglycolate elicitation or endocytosis regardless of ingested materials. From these data, it is concluded that RM-4 recognizes a membrane protein of endolysomes in macrophages and dendritic cells. The antigen may play a role in endolysosomal processing. RM-4 is considered to be a useful tool not only for identifying macrophage/dendritic cells both in frozen and paraffin-embedded tissues, but also for evaluating their endolysosomal processing.

Expression of monocyte chemoattractant protein-1 in experimental crescentic glomerulonephritis in rats.

Crescentic glomerulonephritis (CGN) is a rapidly progressive glomerular disease that is usually associated with a poor prognosis. Monocytes/macrophages are frequently observed in glomeruli in cases of CGN, and they are considered to play a crucial role in the pathogenesis of this disease. In this study we analyzed the glomerular expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for monocytes, in an experimental model of CGN. A model of the disease was induced in the WKY strain of rats by intravenous injection of antiserum raised against glomerular basement membranes. Accumulation of monocytes/macrophages in glomeruli was observed 4 hours after the injection of antiserum. Northern blot analysis showed that the expression of mRNA for MCP-1 was enhanced within 4 hours, peaked on day 3--when it was 60 times that in the control--and then declined. Immunostaining with MCP-1-specific antibody revealed the expression of MCP-1 protein in the diseased glomeruli but not in control glomeruli. Quantitative analysis of glomerular MCP-1 protein by enzyme-linked immunosorbent assay revealed a level 46 times that in the control in reflecting the increase in mRNA for MCP-1. These results indicate that glomeruli of rats with CGN produce MCP-1, which may play an important role in the pathogenesis of glomerular inflammation and crescent formation in CGN.

Pneumocyte biomarkers KL-6 and surfactant protein D reflect the distinct findings of high-resolution computed tomography in nonspecific interstitial pneumonia

Background: Serum levels of pneumocyte biomarkers KL-6 and surfactant protein D (SP-D) are useful diagnostic markers for interstitial lung diseases. However, associations of serum KL-6 and SP-D with radiologic findings in nonspecific interstitial pneumonia (NSIP) remain unclear. Objectives: To determine whether serum levels of KL-6 and SP-D reflect fibrotic and/or inflammatory processes in NSIP, we investigated the correlation between high-resolution computed tomography (HRCT) findings and serum KL-6 and SP-D levels. Methods: Serum levels of KL-6 and SP-D were measured in 21 patients with biopsy-confirmed NSIP. The radiographic extent of 6 HRCT patterns and total HRCT score, defined as the scored fibrotic index, were assessed. Changes in the levels of serum markers and CT findings during follow-up were also monitored. Results: Serum levels of KL-6 in NSIP positively correlated with the total HRCT score and overall extent of interstitial disease. Serum levels of SP-D in NSIP showed a positive correlation with the area of ground-glass attenuation without traction bronchiectasis and the inflammatory CT pattern, but the levels were inversely correlated with the area of ground-glass attenuation with traction bronchiectasis and the fibrotic CT pattern. The follow-up CT and serum marker changes after treatment showed that percent change of disease extent was reflected in both markers, especially KL-6. Further, the decreased fibrotic pattern correlated with both biomarkers. Conclusions: The results indicate that serum levels of KL-6 in NSIP reflect the overall extent of interstitial lesions, which include both inflammatory and fibrotic lesions, while the levels of SP-D mainly reflect the extent of inflammatory lesions.

Production of fibroblast proliferative cytokines from T lymphocytes stimulated by a B cell lymphoma line and their functional heterogeneity

Human mononuclear leukocytes (MNL) produced several factors with fibroblast proliferation activity (FPA) for HFL-1, a human lung fibroblast cell line, when MNL were cocultured with irradiated BALL-1, a B cell lymphoma line (BCLL), but not with other BCLL. The cellular source of BALL-1-induced FPA seemed to be CD4-positive T lymphocytes. On isoelectric electrophoresis, major activity of BALL-1-induced FPA was detected in the fractions around pH 4-5, and minor activity was present in the fractions around pH 6-7. Major BALL-1-induced FPA consisted of at least 4 different fibroblast proliferation factors (FPFs) according to their molecular weight; 320-600 kDa (P-I), 50-110 kDa (P-II), 22-38 kDa (P-III) and 4.6-11 kDa (P-IV). P-I had affinity to heparin though the rest had little or no affinity. FPA of P-I was suppressed by an antibody against acidic FGF, and FPA of P-III was suppressed by an antibody against IL-6. On the other hand, FPA of P-II and P-IV was suppressed by none of the antibodies against cytokines with FPA, such as FGF, IL-4, IL-6, IFN-gamma, TGF-beta and TNF-alpha. It was thus suggested that P-I was acidic FGF, that P-III was IL-6, and that P-II and P-IV were different cytokines from those described above. Furthermore, it was found that P-II and P-IV failed to exhibit proliferation activity for human umbilical vein endothelial cells (HUVEC).(ABSTRACT TRUNCATED AT 250 WORDS)