TLR9 is expressed in idiopathic interstitial pneumonia and its activation promotes in vitro myofibroblast differentiation.

Infectious diseases can be cofactors in idiopathic interstitial pneumonias (IIP) pathogenesis; recent data suggests that toll-like receptors 9 (TLR9) ligands contribute to experimental chronic tissue remodeling. Real-time TAQMAN and immunohistochemical analysis of IIP normal surgical lung biopsies (SLBs), primary fibroblast lines grown from both IIP and normal SLBs indicate that TLR9 is prominently and differentially expressed in a disease-specific manner. TLR9 expression was increased in biopsies from patients with IIP compared with normal lung biopsies and its expression is localized to areas of marked interstitial fibrosis. TLR9 in fibroblasts appeared to be increased by profibrotic Th2 cytokines (IL-4 and IL-13) and this was true in fibroblasts cultured from the most severe form of IIP, idiopathic pulmonary fibrosis (IPF) SLBs, in non-specific interstitial pneumonia fibroblast lines, and in normal fibroblasts. Finally, confocal microscopy studies have shown that TLR9 activation by its synthetic agonist CpG-ODN significantly increased the expression of alpha smooth muscle actin, the main marker of myofibroblast differentiation. These data indicate that TLR9 expression may drive the abnormal tissue healing response in severe forms of IIP and its activation can have a key role in myofibroblast differentiation promoting the progression of disease during the terminal phase of IPF.

Role of interleukin-12 and stat-4 in the regulation of airway inflammation and hyperreactivity in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a respiratory pathogen that can cause significant morbidity in infants and young children. Interestingly, the majority of children who acquire a RSV infection do not exhibit severe symptoms. Development of a Th1 response has been associated with resolution of symptoms in viral infections and may explain mild RSV illness. The current study investigated the cytokine response observed in mild disease in C57BL/6 mice that had low airway resistance and mucus production with little pulmonary inflammation. RSV infection in these mice was accompanied by a fourfold increase in interleukin-12(IL-12). Treatment of RSV-infected mice with anti-IL-12 resulted in an increase in airway hyperreactivity, mucus production, and airway inflammation (eosinophilia). Since IL-12 activation is dependent on Stat-4-mediated intracellular signal transduction, similar experiments were performed in Stat-4 deficient mice and demonstrated similar results to those obtained from anti-IL-12 treated mice. Again, there was an increase in airway hyperreactivity and mucus production, and goblet cell hypertrophy. These studies support the importance of IL-12 in the immune response to RSV infection resulting in resolution of disease and protection from inappropriate inflammatory responses.

Pivotal role of the CC chemokine, macrophage-derived chemokine, in the innate immune response.

Macrophage-derived chemokine (MDC), a recently identified CC chemokine, has been regarded to be involved in chronic inflammation and dendritic cell and lymphocyte homing. In this study, we demonstrate a pivotal role for MDC during experimental sepsis induced by cecal ligation and puncture (CLP). Intraperitoneal administration of MDC (1 microg/mouse) protected mice from CLP-induced lethality. The survival was accompanied by increased number of peritoneal macrophages and decreased recovery of viable bacteria from the peritoneum and peripheral blood. In addition, mice treated with an i.p. injection of MDC cleared bacteria more effectively than those in the control when 3 x 108 CFU live Escherichia coli was i.p. inoculated. Endogenous MDC was detected in the peritoneum after CLP, and neutralization of the MDC with anti-MDC Abs decreased CLP-induced recruitment of peritoneal macrophages and increased the recovery of viable bacteria from the peritoneum and peripheral blood. MDC blockade was deleterious in the survival of mice after CLP. In vitro, MDC enhanced the phagocytic and killing activities of peritoneal macrophages to E. coli and induced both a respiratory burst and the release of lysozomal enzyme from macrophages. Furthermore, MDC dramatically ameliorated CLP-induced systemic tissue inflammation as well as tissue dysfunction, which were associated in part with decreased levels of TNF-alpha, macrophage inflammatory proteins-1alpha and -2, and KC in specific tissues. Collectively, these results indicate novel regulatory activities of MDC in innate immunity during sepsis and suggest that MDC may aid in an adjunct therapy in sepsis.

Expression and contribution of endogenous IL-13 in an experimental model of sepsis.

IL-13 has been shown to exert potent anti-inflammatory properties. In this study, we elucidated the functional role of endogenous IL-13 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that the level of IL-13 increased in tissues including liver, lung, and kidney, whereas no considerable increase was found in either peritoneal fluid or serum after CLP. Immunohistochemically, IL-13-positive cells were Kupffer cells in liver, alveolar macrophages in lung, and epithelial cells of urinary tubules in kidney. IL-13 blockade with anti-IL-13 Abs significantly decreased the survival rate of mice after CLP from 53% to 14% on day 7 compared with control. To determine the potential mechanisms whereby IL-13 exerted a protective role in this model, the effects of anti-IL-13 Abs on both local and systemic inflammation were investigated. Administration of anti-IL-13 Abs did not alter the leukocyte infiltration and bacterial load in the peritoneum after CLP but dramatically increased the neutrophil influx in tissues after CLP, an effect that was accompanied by significant increases in the serum levels of aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine. Tissue injury caused by IL-13 blockade was associated with increases in mRNA and the protein levels of CXC chemokines macrophage inflammatory protein-2 and KC as well as the CC chemokine macrophage inflammatory protein-1alpha and the proinflammatory cytokine TNF-alpha. Collectively, these results suggest that endogenous IL-13 protected mice from CLP-induced lethality by modulating inflammatory responses via suppression of overzealous production of inflammatory cytokines/chemokines in tissues.

Mast cells produce ENA-78, which can function as a potent neutrophil chemoattractant during allergic airway inflammation.

The inflammatory response during allergic airway inflammation involves the recruitment of multiple leukocyte populations, including neutrophils, monocytes, lymphocytes, and eosinophils. All of these populations likely contribute to the pathology observed during repeated episodes of allergic airway inflammation. We have examined the role of a human neutrophil-specific chemokine (C-x-C), ENA-78, in a model of allergic airway responses and identified murine mast cells as a cellular source of an ENA-78-like molecule. Within this allergic airway model, neutrophil infiltration into the airway occurs within 4-8 h post-allergen challenge, persists within the airway until 24 h, and resolves by 48 h post-challenge. Neutrophil influx precedes the eosinophil infiltration, which peaks in the airway at 48 h post-allergen challenge. In this study the production of ENA-78 from challenged lungs demonstrated a significant increase in the allergen-, but not vehicle-, challenged lungs. In vivo neutralization of ENA-78 by passive immunization demonstrated a significant decrease in peak neutrophil infiltration at 8 h, with no effect on the eosinophil infiltration at 48 h post-challenge. Because ENA-78 has been shown to be chemotactic for neutrophils and given the involvement of mast cell degranulation in allergic responses, we examined mast cells for the presence of ENA-78. Cultured mast cells spontaneously released ENA-78, but on activation with IgE + antigen, NG-L-arginine methyl ester or compound 48/80 produced significantly increased levels of ENA-78. Supernatants from sonicated MC-9 mast cells induced an overwhelming influx of neutrophils into the BAL by 4 h post-intratracheal injection into mice, suggesting that the mast cell is a significant source of neutrophil chemotactic factors. Mast cell supernatant-mediated neutrophil infiltration was substantially decreased by preincubation of the supernatant with antibodies specific for ENA-78. These data indicate a major neutrophil chemotactic protein produced by mast cells during allergic responses may be mast cell-derived ENA-78.

The role of stem cell factor (c-kit ligand) and inflammatory cytokines in pulmonary mast cell activation.

Mast cells play a critical role in allergic airway responses via IgE-specific activation and release of potent inflammatory mediators. In the present study, we have isolated and characterized primary mast cell lines derived from the upper airways of normal mice. The primary mast cell lines were grown and maintained by incubation with interleukin-3 (IL-3) and stem cell factor (SCF) and shown to be c-kit (SCF receptor) positive by flow cytometry. Subsequently, we examined the proliferation of both airway and bone marrow derived mast cell lines in response to inflammatory and hematopoietic cytokines, including SCF, IL-1, IL-3, interferon-gamma, IL-4, and IL-10. The results from the pulmonary mast cell lines were compared with those from bone marrow derived mast cells. Pulmonary mast cell lines were capable of proliferating in response to IL-3, IL-4, IL-10, and SCF, whereas the combination of SCF with the other cytokines did not increase the response over SCF alone. In contrast, the bone marrow-derived mast cells proliferated strongest to SCF or IL-3, but only modestly to IL-4 and IL-10. Furthermore, the combination of SCF with IL-3, but not the other cytokines, exhibited an increase in bone marrow-derived mast cell proliferation. Cytokine-specific stimulation of histamine release in the airway-derived and bone marrow-derived mast cells showed parallel results. SCF was the only cytokine shown to induce substantial histamine release. However, when certain nonhistamine releasing cytokines were combined with SCF, a synergistic increase in histamine release was induced in upper airway, but not bone marrow-derived mast cells. The results of these studies suggest that cytokines differentially modulate induction of proliferation and degranulation of bone marrow and upper airway-derived mast cells and may further indicate a cytokine activational cascade in tissue mast cells.

TNF-induced IL-8 and MCP-1 production in the eosinophilic cell line, EOL-1.

The role of eosinophils in inflammation and their mode of activation is not well understood. Eosinophil accumulation and subsequent expression of cytokines at the site of inflammation may play a role in exacerbation of inflammatory responses. In the present study, we have examined the role of TNF-alpha in eosinophil activation and chemokine production using a human leukaemic eosinophil cell line, EOL-1. Initial studies demonstrated that TNF-alpha induced the upregulation of IL-8 and MCP-1 mRNA and protein. Kinetic studies indicated production of chemokines, IL-8 and MCP-1, as early as 4 h post-activation, with peak levels of chemokine produced at 8 h, and decreasing by 24 h post-TNF-alpha activation. When IL-10, a suppressive cytokine, was incubated with TNF-alpha and EOL-1 cells, no effect was observed on IL-8 and MCP-1 production. However, dexamethasone, a glucocorticoid, demonstrated potent inhibitory effects on the EOL-1-derived chemokines. These studies indicate that eosinophils may be a significant source of chemokines capable of participating in, and maintaining, leukocyte recruitment during inflammatory responses, such as asthma.

Production of chemokines, interleukin-8 and monocyte chemoattractant protein-1, during monocyte: endothelial cell interactions.

The extravasation of leukocytes from the lumen of the vessel to a site of inflammation requires specific binding events. The interaction of leukocytes with endothelium, via specific receptors, may provide intracellular signals that activate extravasating cells. In the present study, we have investigated the production of chemokines, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) during monocyte: endothelial cell interactions. Both unstimulated and interferon-gamma (IFN-gamma)-prestimulated human umbilical vein endothelial cells (HUVEC) produced low constitutive levels of IL-8 and MCP-1. The addition of enriched monocytes with unstimulated HUVEC resulted in synergistic increases in production of both IL-8 and MCP-1. Monocytes cultured with IFN-gamma-preactivated HUVECs demonstrated little additional increase in IL-8 and MCP-1 production in coculture assays compared with unstimulated HUVEC. Northern blot analysis paralleled the protein data, demonstrating upregulated expression of IL-8 and MCP-1 mRNA in stimulated and unstimulated coculture assays. Culture of enriched monocytes and endothelial cells in transwells demonstrated no increases in IL-8 or MCP-1, indicating the necessity for cellular contact for chemokine production. In previous investigations, we have demonstrated that increased monocyte-derived MIP-1 alpha production was induced by intracellular adhesion molecule-1 (ICAM-1) interactions on activated HUVECs. In contrast, addition of anti-ICAM-1 monoclonal antibodies (MoAbs) did not diminish the production of IL-8 and MCP-1 in the present study. Furthermore, neither antibodies to IL-1 nor tumor necrosis factor (TNF) diminished the production of either IL-8 or MCP-1. However, when soluble matrix proteins were added to the coculture to block cellular interactions, the chemokine protein and mRNA levels were significantly decreased. IL-8 production was decreased by both soluble collagen and fibronectin, whereas MCP-1 was decreased by only soluble collagen, suggesting differential activation pathways. These results indicate that IL-8 and MCP-1 production are increased during monocyte and endothelial cell interactions in part due to matrix protein binding mechanisms. This mechanism may serve a role in cell activation, production of chemokines, as well as extravasation and recruitment of additional leukocytes during inflammatory responses.

Stimulus and cell-specific expression of C-X-C and C-C chemokines by pulmonary stromal cell populations.

Chronic inflammatory responses in the lung rely on the continual recruitment of leukocytes to the site of inflammation. Recent data have demonstrated a possible role for stromal cell-derived chemokines in leukocyte recruitment. In the present study we examined the production of interleukin (IL)-8 and ENA-78, members of the C-X-C family of chemokines, and macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, members of the C-C chemokine family, from pulmonary smooth muscle and endothelial cells. The production of IL-8 and ENA-78 was induced by early response cytokines, IL-1 and tumor necrosis factor (TNF), but not by immune-associated cytokines, IL-4, IL-10, or interferon (IFN)-gamma. In contrast, the production of MIP-1 alpha and MIP-1 beta by pulmonary vascular smooth muscle cells increased when stimulated by immune-associated cytokines as well as with IL-1 beta and TNF. The level of MIP-1 alpha production induced in smooth muscle cells by the immune-associated cytokines, IL-4, IFN-gamma, and IL-10 ranged from 0 to 340 pg/ml. The production of MIP-1 beta in response to the immune-associated cytokines IL-4, IFN-gamma, and IL-10 in smooth muscle cells ranged from 260 to 940 pg/ml. Human pulmonary artery endothelial cells did not generate MIP-1 alpha or MIP-1 beta in response to graded doses of any of the cytokines. These data demonstrate differential induction of C-X-C and C-C chemokines from nonimmune stromal cell populations.(ABSTRACT TRUNCATED AT 250 WORDS)

VCAM-1 influences lymphocyte proliferation and cytokine production during mixed lymphocyte responses.

The allogeneic mixed lymphocyte reaction (MLR) is regarded as an effective model for examining the events which occur during an allospecific immune response. Numerous studies have delineated the role of adherence molecule interactions during the MLR response. In the present study we have identified VCAM-1 as having a contribution to the generation of an allogeneic MLR response. These findings may have broad implications in vivo during antigen-specific and allograft rejection events. RT-PCR analysis was initially used to examine whether VCAM-1 mRNA expression was observed during MLR responses and demonstrated peak expression between 12 and 48 hr of culture. Immunolocalization of VCAM-1 on adherent mononuclear phagocytes, but not nonadherent lymphocytes, from MLR cultures verified its expression during this response. Addition of anti-VCAM-1 mAbs to MLR assays inhibited the proliferative response by over 70%, while addition of anti-VCAM-1 as late as Day 2 of the assay allowed significant inhibition of the proliferative response. This correlated with peak expression of VCAM-1 mRNA observed as late as 48 hr in RT-PCR analyses. In further studies, anti-VCAM-1 significantly inhibited peak expression of IL-2 on Days 3 and 4, while TNF-alpha production was diminished at 30 min and 1, 96, and 120 hr of culture, compared to control cultures. The production of macrophage inflammatory protein-1 alpha (MIP-1 alpha), a chemotactic cytokine which has an important role in vivo for the recruitment of leukocytes to a site of inflammation, was also significantly inhibited during peak production on Days 4 and 5 of the MLR assay. This study demonstrates novel findings of VCAM-1 expression during an allogeneic MLR response. The expression of VCAM-1 may have important implications during allospecific immune responses for the activation and proliferation of T lymphocytes as well as cytokine production.

Intercellular adhesion molecule-1 mediates the expression of monocyte-derived MIP-1 alpha during monocyte-endothelial cell interactions.

The extravasation of leukocytes from the lumen of the vessel to a site of inflammation initially requires a specific binding event followed by migration of the cells through the endothelial cell layer into the inflammatory foci. The interaction of leukocytes with the endothelium via specific receptors may provide intracellular signals that activate the cells. In the present study we have investigated the production of MIP-1 alpha, a mononuclear cell chemotactic protein, during monocyte:endothelial cell interactions. Neither unstimulated nor interferon (IFN)-stimulated human umbilical vein endothelial cells (HUVECs) produced substantial MIP-1 alpha protein. However, the addition of enriched monocyte populations with unstimulated HUVECs resulted in the production of MIP-1 alpha. Monocytes cultured with IFN-gamma-activated HUVECs showed an additional increase in MIP-1 alpha production. Immunohistochemical analysis demonstrated that the monocyte was the cellular source of MIP-1 alpha production in this coculture system. The mechanism of MIP-1 alpha expression was further assessed by determining the role of adhesion molecules in the regulation of MIP-1 alpha production during monocyte:HUVEC interactions. To attenuate the increased production of MIP-1 alpha by the monocyte:HUVEC interaction, anti-adhesion molecule monoclonal antibodies (MoAbs) were added to the cultures. Addition of anti-ICAM-1 neutralizing MoAbs significantly inhibited the production of MIP-1 alpha, whereas neutralizing anti-VCAM-1 MoAbs failed to block MIP-1 alpha production. Furthermore, MIP-1 alpha production was induced in monocytes cultured on ICAM-1-coated plates. These results indicate an intimate relationship between leukocyte-endothelial cells, adhesion molecule, and the expression of the monocyte-derived chemokine MIP-1 alpha during cellular adhesion. This mechanism may serve an important role in cell activation and recruitment of leukocytes during the initiation of an inflammatory response.

Enhanced production of the chemotactic cytokines interleukin-8 and monocyte chemoattractant protein-1 in human abdominal aortic aneurysms.

Inflammatory leukocytes play a central role in the pathogenesis of human atherosclerotic disease, from early atherogenesis to the late stages of atherosclerosis, such as aneurysm formation. We have shown previously that human abdominal aortic aneurysms are characterized by the presence of numerous chronic inflammatory cells throughout the vessel wall (Am J Pathol 1990, 137: 1199-1213). The signals that attract lymphocytes and monocytes into the aortic wall in aneurysmal disease remain to be precisely defined. We have studied the production of the chemotactic cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by aortic tissues obtained from 47 subjects. We compared the antigenic production of these cytokines by explants of: 1) human abdominal aneurysmal tissue, 2) occlusive (atherosclerotic) aortas, and 3) normal aortas. IL-8, which is chemotactic for neutrophils, lymphocytes, and endothelial cells was liberated in greater quantities by abdominal aortic aneurysms than by occlusive or normal aortas. Using immunohistochemistry, macrophages, and to a lesser degree endothelial cells, were found to be positive for the expression of antigenic IL-8. Similarly, MCP-1, a potent chemotactic cytokine for monocytes/macrophages, was released by explants from abdominal aortic aneurysms in greater quantities than by explants from occlusive or normal aortas. Using immunohistochemistry, the predominant MCP-1 antigen-positive cells were macrophages and to a lesser extent smooth muscle cells. Our results indicate that human abdominal aortic aneurysms produce IL-8 and MCP-1, both of which may serve to recruit additional inflammatory cells into the abdominal aortic wall, hence perpetuating the inflammatory reaction that may result in the pathology of vessel wall destruction and aortic aneurysm formation.

Expression and regulation of human pulmonary fibroblast-derived monocyte chemotactic peptide-1.

Monocyte recruitment is essential for maintenance of normal pulmonary macrophage populations. In addition, acute and chronic inflammatory pulmonary diseases are associated with sequestration of mononuclear phagocytes in the lung. Although alveolar macrophages (AM phi) can secrete a number of potent inflammatory and chemoattractment mediators, these immune cells do not produce monocyte chemotactic peptide (MCP-1) in response to lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin-1 beta (IL-1 beta). The pulmonary fibroblast (PF) may play a pivotal role in monocyte recruitment. In these studies, we demonstrate a time- and dose-dependent production of PF-derived steady-state MCP-1 mRNA, MCP-1 antigen, and monocyte chemotactic bioactivity attributable to MCP-1. In cellular models examining cytokine networks between AM phi and PF, LSP-stimulated AM phi (conditioned media) induced PF-derived steady-state MCP-1 mRNA expression that was markedly attenuated by the presence of neutralizing TNF and IL-1 beta antibodies. Furthermore, we showed the dose- and time-dependent suppression of IL-1 beta-stimulated PF-derived MCP-1 by dexamethasone and prostaglandin E2. These findings demonstrated that PF are an important cellular source of MCP-1 and this production of MCP-1 may be influenced by immunomodulators.

Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis.

Cells within the synovial tissue may recruit mononuclear phagocytes into the synovial fluid and tissues of arthritic patients. We investigated the production of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1) using sera, synovial fluid, synovial tissue, as well as macrophages and fibroblasts isolated from synovial tissues from 80 arthritic patients. MCP-1 levels were significantly higher (P less than 0.05) in synovial fluid from RA patients (mean 25.5 +/- 8.1 ng/ml [SE]) compared to synovial fluid from osteoarthritis (OA) patients (0.92 +/- 0.08), or from patients with other arthritides (2.9 +/- 1.5). MCP-1 levels in RA sera (8.44 +/- 2.33) were significantly greater than MCP-1 in normal sera (0.16 +/- 0.06). The quantities of RA synovial fluid IL-8, which is chemotactic for neutrophils and lymphocytes, and MCP-1 were strongly positively correlated (P less than 0.05). To examine the cellular source of MCP-1, RA synovial tissue macrophages and fibroblasts were isolated. Synovial tissue fibroblasts did not express MCP-1 mRNA, but could be induced to produce MCP-1 by stimulation with either IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), or LPS. In contrast, unlike normal peripheral blood monocytes or alveolar macrophages, RA synovial tissue macrophages constitutively expressed MCP-1 mRNA and antigen. Immunohistochemical analysis of synovial tissue showed that a significantly greater percentage of RA macrophages (50 +/- 8%) as compared to either OA macrophages (5 +/- 2) or normal macrophages (1 +/- 0.3) reacted with anti-MCP-1 antibodies. In addition, the synovial lining layer reacted with MCP-1 in both RA and OA synovial tissues. In contrast, only a minority of synovial fibroblasts (18 +/- 8%) from RA synovium were positive for immunolocalization of MCP-1. These results suggest that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with RA and that synovial tissue macrophages are the dominant source of this cytokine.

A sensitive ELISA for the detection of human monocyte chemoattractant protein-1 (MCP-1).

The recruitment of monocytes into tissue is associated with both acute and chronic inflammation. Although monocyte migration is measured in vitro by monocyte chemotaxis, this technique is often difficult to determine the specific quantitative contribution of a monocyte chemotaxin. We have developed a sensitive sandwich ELISA for the detection of monocyte chemoattractant protein-1 (MCP-1), a highly specific monocyte activating/chemotactic peptide. Polyclonal antibodies were generated from rabbits. The IgG fraction of the antiserum was isolated by a protein A column, with a portion of the antibodies biotinylated. Avidin-conjugated horse radish peroxidase was used for enzymatic, colorimetric analysis. The lower threshold for detection of MCP-1 was 50 pg/ml, and the ELISA was specific for MCP-1, since it failed to recognize other cytokines in a dose-dependent fashion. Furthermore, this ELISA had the capacity to measure endothelial cell and pulmonary fibroblast-derived MCP-1. The development of a sensitive ELISA for the detection MCP-1 is significant, since it will allow the measurement MCP-1 from biologically relevant fluids, and aid in establishing whether MCP-1 is present in disease.

Role of IL-4 and IFN-gamma in Schistosoma mansoni egg-induced hypersensitivity granuloma formation. Orchestration, relative contribution, and relationship to macrophage function.

A well defined model of T cell-mediated hypersensitivity-type granulomatous inflammation induced by Schistosoma mansoni eggs was used to assess the role of IL-4 and IFN-gamma in granuloma development. Synchronized pulmonary granulomas were induced and isolated from S. mansoni-infected mice during vigorous (8 wk) and modulated (20 wk) stages of the disease. The sequential production of IL-4 and IFN was determined and related to temporal changes in granuloma macrophage production of IL-1, TNF, and superoxide anion (O2-). During the vigorous stage, IL-4 was produced on days 1 and 2 of granuloma formation, whereas IFN was released in greatest amounts on days 4 to 8. The peak of IL-4 occurred in a window between the peak of IL-1 (1 day) and maximal TNF production (8 to 16 days). Maximal O2- release tended to parallel IFN production. During the modulated stage when the inflammatory response is attenuated, IL-4 production was dramatically reduced as were levels of IL-1 and TNF, but IFN production persisted and maximum O2(-)-producing capacity was only delayed in onset. mAb specific for IL-4 and IFN were used to examine the effect of in vivo depletion of these cytokines on granuloma development. Administration of a single 1.0-mg dose of anti-IL-4 antibodies to mice with synchronously developing granulomas dramatically reduced granuloma size (40 to 50% suppression of area) during an 8-day study period, whereas antibodies to IFN had no effect on size. However, the latter treatment reduced giant cell formation. Our results indicate that granuloma development involves an orchestrated production of cytokines possibly resulting from sequential participation of different Th cell populations. Moreover, IL-4 is a pivotal cytokine in anamnestic cellular recruitment and subject to endogenous regulation.

Regulation of human alveolar macrophage- and blood monocyte-derived interleukin-8 by prostaglandin E2 and dexamethasone.

Mononuclear phagocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities, monocytes/macrophages produce a vast array of regulatory and chemotactic cytokines. Interleukin-8 (IL-8), a potent neutrophil-activating and chemotactic peptide, is produced in large quantities by mononuclear phagocytes and may be an important mediator of local and systemic inflammatory events. In this investigation, we describe the effects of prostaglandin E2 (PGE2) and dexamethasone (Dex) on IL-8 mRNA and protein expression from lipopolysaccharide (LPS)-treated human peripheral blood monocytes (PBM) and alveolar macrophages (AM). We demonstrate the dose-dependent suppression of IL-8 from LPS-stimulated PBM by PGE2. Treatment of stimulated PBM with 10(-6) M PGE2 resulted in maximal inhibition, causing 60% suppression of both IL-8 mRNA and extracellular protein levels. In contrast, PGE2 (10(-6) to 10(-8) M) did not significantly alter IL-8 mRNA or protein expression from LPS-treated AM. Treatment of LPS-stimulated PBM and AM with Dex (10(-6) to 10(-8) M) resulted in 75% decline in IL-8 mRNA and extracellular protein from either cell population. Pretreatment of PBM with PGE2 or Dex 1 or 2 h before LPS stimulation caused a significant suppression of steady-state IL-8 mRNA levels; however, administration of either of these modulators 1 or 2 h after LPS stimulation failed to have an inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary fibroblast expression of interleukin-8: a model for alveolar macrophage-derived cytokine networking.

The pulmonary fibroblast's (PF) unique location allows it to communicate in a bidirectional fashion between the vascular compartment and alveolar airspace, placing it in a strategic position for the elicitation of inflammatory leukocytes into the lung. In this study, we demonstrate that PF may contribute to pulmonary inflammation through the production of a potent neutrophil chemotactic factor, interleukin (IL)-8. PF-derived IL-8 expression was dependent upon stimulation by either tumor necrosis factor (TNF) or IL-1 but not lipopolysaccharide (LPS). Both TNF and IL-1 stimulation of PF resulted in a time- and dose-dependent expression of steady-state levels of mRNA, antigen, and specific chemotactic activity consistent with IL-8. Because it was apparent that cytokine networking may exist in the lung between alveolar macrophage (AM)-derived cytokines and the production of PF-derived IL-8, we next examined an in vitro model of cellular communication within the lung. We determined that LPS-stimulated AM-conditioned media induced significant levels of PF-derived IL-8 mRNA, which was inhibited by preincubation with specific neutralizing TNF and IL-1 beta antibodies. Furthermore, when AM were directly co-cultured with PF and stimulated with LPS, the kinetic analysis of PF-derived antigenic expression of IL-8 was shifted toward the right. This suggested that PF-derived IL-8 expression in co-culture was first dependent upon activation of the AM by LPS and subsequent elaboration of macrophage inflammatory mediators. These data provide evidence that cytokine networking between AM and PF may be operative in the lung, culminating in the generation of IL-8 and elicitation of inflammatory leukocytes.

Synovial tissue macrophage as a source of the chemotactic cytokine IL-8.

Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.