Attenuation of lung inflammation and fibrosis in interferon-gamma-deficient mice after intratracheal bleomycin.

Because mouse strains susceptible to bleomycin, such as C57BL/ 6J, tend to produce T helper type 1 (Th1) cytokines in response to immune activation, we hypothesized that the inflammatory response to bleomycin is mediated, in part, by local production of the Th1 cytokine interferon-gamma (IFN-gamma). Consistent with this hypothesis, fibrosis-prone C57BL/6J and A/J mice demonstrated significantly elevated expression of IFN-gamma protein (by enzyme-linked immunosorbent assay) in bronchoalveolar lavage fluid at 24 h, and subsequently increased lung inflammation, weight loss, and mortality 10 d after intratracheal bleomycin administration compared with fibrosis-resistant BALB/c mice or saline control mice. To directly determine a role for IFN-gamma in bleomycin toxicity, we exposed C57BL/6J mice with a homozygous null mutation of the IFN-gamma gene (IFN-gamma[-/-]) and wild-type C57BL/6J mice to intratracheal bleomycin. IFN-gamma(-/-) mice demonstrated significantly lower parenchymal inflammation, weight loss, and mortality 10 d after 5 U/kg intratracheal bleomycin administration compared with control mice. At 3 wk after 1.5 U/kg bleomycin exposure, single lung collagen determined by hydroxyproline assay was significantly lower in IFN-gamma(-/-) mice compared with wild-type C57BL/6J mice. Together, these results suggest that IFN-gamma mediates, in part, bleomycin-induced pulmonary inflammation and fibrosis.

Inhibition of IL-12 production by thalidomide.

The immunomodulatory properties of thalidomide are currently being exploited therapeutically in conditions as diverse as erythema nodosum leprosum, chronic graft-vs-host disease, rheumatoid arthritis, and sarcoidosis. The relevant mechanism of action of thalidomide in these diseases remains unclear. The important role recently ascribed to IL-12, a cytokine critical to the development of cellular immune responses, in the pathogenesis of several of these conditions led us to examine whether thalidomide affects the production of IL-12. Thalidomide potently suppressed the production of IL-12 from human PBMC and primary human monocytes in a concentration-dependent manner. Thalidomide-induced inhibition of IL-12 production was additive to that induced by suboptimal inhibiting doses of dexamethasone, and occurred by a mechanism independent of known endogenous inhibitors of IL-12 production. These results suggest that thalidomide may have therapeutic utility in a wide range of immunologic disorders that are characterized by inappropriate cellular immune responses.

Inhibition of human interleukin-12 production by pentoxifylline.

Pharmacological control of interleukin-12 (IL-12) production may be a key therapeutic strategy for modulating immunological diseases dominated by type-1 cytokine responses. In this study, we investigated the effects of pentoxifylline on the production of IL-12 by human blood mononuclear cells and primary human monocytes stimulated with heat-killed Staphylococcus aureus Cowan strain I (SAC) or lipopolysaccharide (LPS). Pentoxifylline potently suppressed production of IL-12 in a concentration-dependent manner. In these same experiments, tumour necrosis factor-alpha (TNF-alpha) production was inhibited and IL-10 and prostaglandin E2 (PGE2) production was enhanced by treatment with pentoxifylline. Suppression of IL-12 production by pentoxifylline was found to be independent of several known endogenous inhibitors of IL-12, such as IL-10, transforming growth factor-beta (TGF-beta), IL-4 and PGE2. RNase protection assays revealed that pentoxifylline inhibited accumulation of both IL-12 p40 and p35 mRNA, suggesting a predominant mRNA locus for pentoxifylline-induced IL-12 inhibition. Low levels of pentoxifylline added to the suppression of IL-12 production by suboptimal inhibiting doses of dexamethasone, suggesting that this drug combination may have therapeutic utility. These results provide a firm rationale for the use of pentoxifylline in clinical trials of immunological disorders characterized by inappropriate type-1 immune responses.

Enhanced expression of IL-12 associated with Th1 cytokine profiles in active pulmonary sarcoidosis.

Sarcoidosis is a multisystem granulomatous disease of unknown etiology characterized by the expansion of activated oligoclonal CD4+ T cells and macrophages at sites of disease. To investigate the immunopathogenesis of sarcoidosis, we analyzed patterns of cytokine expression in bronchoalveolar lavage cells and fluid from patients with pulmonary sarcoidosis and idiopathic pulmonary fibrosis and from normal volunteers. We found dominant type 1 cytokine expression, with elevated mRNA and protein levels of IFN-gamma, but not IL-4, in sarcoid lung cells and fluid compared with those in normal samples. To define immunoregulatory mechanisms important to this type 1 response, we analyzed the expression of IL-12 and IL-10 in lung cells and fluid. Using semiquantitative PCR, we found significantly higher mRNA expression of the regulated IL-12 p40 subunit, but not IL-10, in sarcoid compared with normal lung cells. Consistent with these observations, strikingly elevated levels of p40 protein were found in sarcoid compared with normal bronchoalveolar lavage fluid. Unstimulated and Staphylococcus aureus-stimulated sarcoid alveolar macrophages produced greater amounts of IL-12 than normal alveolar macrophages when cultured in vitro. We hypothesize that sarcoidosis is a Th1-mediated disease driven by chronic, dysregulated production of IL-12 at sites of disease.

Selective activation and accumulation of oligoclonal V beta-specific T cells in active pulmonary sarcoidosis.

Sarcoidosis is a granulomatous disease in which activated T cells, responding to an unidentified stimulus, accumulate at sites of disease such as the lung. To evaluate the hypothesis that active sarcoidosis is characterized by a selective activation and expansion of a limited repertoire of T cell receptor (TCR) specific T cells, we analyzed TCR V beta gene expression in lung and blood T cells of patients with active sarcoidosis and, for comparison, normal individuals using polymerase chain reaction amplification of 20 V beta gene families. Analysis of normal bronchoalveolar lavage T cells revealed TCR V beta distributions similar to that of normal blood, providing evidence for a lack of generalized skewing of the T cell repertoire in the normal, noninfected lung. Compared to normal lung and blood, subgroups of individuals with sarcoidosis demonstrated biased expression of one or more V beta genes in either the lung or blood. Five V beta gene families (V beta 5, V beta 8, V beta 15, V beta 16, and V beta 18) were most frequently utilized in a biased fashion by sarcoid lung or blood T cells. Furthermore, dramatic skewing of the T cell repertoire was apparent when sarcoid lung and blood T cells were expanded by short-term culture with IL-2. Sequence analysis demonstrated a bias in V beta gene expression was usually due to expansion of select V beta-specific clones, some of which contained a similar V(D)J junctional region motif. These observations provide evidence for a selective activation and accumulation of antigen-specific V beta-expressing T cells in sarcoidosis.

IgE-dependent histamine-releasing factors. A brief review.

A cytokine, termed histamine-releasing factor (HRF) and produced by many cell types, has become the focus of research by many investigators due to its potential importance as a stimulus in chronic inflammation. We are producing and characterizing an HRF which causes IgE-mediated histamine release from human basophils. Following extensive purification procedures, the molecule will be sequenced and synthesized. A functional heterogeneity of IgE molecules was revealed by these studies. We are currently producing IgE antibody in vitro and testing the hypothesis that differential glycosylation is the basis for the heterogeneity. Knowledge of the structures and interactions of these molecules should advance our understanding of allergic and more chronic diseases.