Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo.

Resistance to chemotherapy is a principal problem in the treatment of small cell lung cancer (SCLC). We show here that SCLC is surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites. Adhesion of SCLC cells to ECM enhances tumorigenicity and confers resistance to chemotherapeutic agents as a result of beta1 integrin-stimulated tyrosine kinase activation suppressing chemotherapy-induced apoptosis. SCLC may create a specialized microenvironment, and the survival of cells bound to ECM could explain the partial responses and local recurrence of SCLC often seen clinically after chemotherapy. Strategies based on blocking beta1 integrin-mediated survival signals may represent a new therapeutic approach to improve the response to chemotherapy in SCLC.

Expression and regulation of human neutrophil-derived macrophage inflammatory protein 1 alpha.

Neutrophil (polymorphonuclear leukocyte [PMN]) sequestration is one of the histologic hallmarks of an acute inflammatory response. During the natural evolution of an inflammatory response, PMNs are often replaced by mononuclear cells. This shift in the elicitation of specific leukocyte populations usually occurs as the inflammatory lesion enters either the repair/resolution stage or progresses to a chronic inflammation. To elucidate a potential mechanism for the temporal change from predominantly PMN recruitment to the presence of monocytes, we postulated that PMNs could be a rich source of monocyte chemotactic factors. In our studies, we have identified a dose-dependent induction of monocyte chemotactic activity by PMNs treated with lipopolysaccharide (LPS; 1-100 ng/ml). Interestingly, this monocyte chemotactic activity was significantly attenuated in the presence of neutralizing anti-human macrophage inflammatory protein 1 alpha (MIP-1 alpha) antibodies. Moreover, immunolocalization studies demonstrated the expression of MIP-1 alpha by stimulated PMNs. These findings showed that a significant amount of PMN-derived monocyte chemotactic activity was attributable to MIP-1 alpha. Subsequent characterization of MIP-1 alpha steady-state mRNA and antigen expression demonstrated both a dose- and time-dependent production by LPS-treated PMNs. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a potent PMN activator, failed to induce the expression of MIP-1 alpha over a wide range of concentrations. However, PMNs stimulated in the presence of both LPS and GM-CSF resulted in a synergistic expression pattern for MIP-1 alpha. PMNs stimulated in the presence of both GM-CSF and LPS demonstrated an enhanced and prolonged expression for both MIP-1 alpha mRNA and antigen, as compared with LPS alone. Messenger RNA stabilization analyses demonstrated that MIP-1 alpha mRNA isolated from PMNs stimulated in the presence of GM-CSF and LPS had a prolonged mRNA t1/2, as compared with LPS alone. These findings support the notion that PMNs are capable of producing MIP-1 alpha in the presence of LPS, and that GM-CSF can influence this production through prolongation of MIP-1 alpha mRNA t1/2. The production of PMN-derived MIP-1 alpha, in association with the expression of appropriate adhesion molecules at a site of inflammation, may be one of the central events that contributes to the temporal shift from predominantly PMNs to monocytes during the evolution of inflammation.

The role of macrophage inflammatory protein 1 alpha in Schistosoma mansoni egg-induced granulomatous inflammation.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a 6-8-kD, lipopolysaccharide-inducible monocyte and neutrophil chemotactic protein that may be important in acute and chronic inflammation. The present study determined the sequential production, source, and in vivo contribution of murine MIP-1 alpha in synchronized Schistosoma mansoni egg pulmonary granuloma formation. Granulomas were examined under conditions of primary, secondary vigorous, and secondary immunomodulated immunity. Secreted MIP-1 alpha was measured in 24-h supernatants from intact granulomas (700/ml) cultured with or without soluble egg antigen (SEA). Primary granulomas isolated from naive mice over a 16-d period showed low spontaneous MIP-1 alpha production (< 1 ng/ml). However, when primary granulomas were challenged with SEA, significant MIP-1 alpha production was observed beginning at day 4 and peaking at day 16. Intact vigorous (isolated from 8-wk-infected mice) and modulated (isolated from 20-wk-infected mice) secondary pulmonary granulomas demonstrated comparable spontaneous MIP-1 alpha production. Addition of SEA to vigorous stage granulomas augmented expression of MIP-1 alpha at all time points, whereas stimulated modulated stage granulomas did not increase production. The latter observation is likely related to endogenous immunoregulatory mechanisms reported for modulated stage animals. Immunohistochemical localization of MIP-1 alpha in granuloma sections and cytocentrifuge preparations from vigorous lesions localized MIP-1 alpha production to macrophages within granulomas. Treatment of mice with rabbit anti-mouse MIP-1 alpha antibodies significantly decreased 8-d primary granuloma formation (> 40%) when compared with control mice. Anti-MIP-1 alpha sera also decreased vigorous (> 20%), but not modulated granuloma formation. These findings demonstrate that MIP-1 alpha contributes to cellular recruitment during schistosome egg granuloma formation.

Structure and neutrophil-activating properties of a novel inflammatory peptide (ENA-78) with homology to interleukin 8.

A new neutrophil-activating peptide, termed ENA-78, was identified in the conditioned media of stimulated human type II epithelial cell line A549. In response to stimulation with either interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha), ENA-78 was produced and secreted concomitantly with IL-8, GRO alpha, and GRO gamma. ENA-78 consists of 78 amino acids [sequence; see text] and has a molecular weight of 8,357. It has four cysteines positioned identically to those of IL-8 and analogues, and thus belongs to the CXC family of peptides. ENA-78 is related to neutrophil-activating peptide 2 (NAP-2) and GRO alpha (sequence identity, 53% and 52%, respectively) and IL-8 (22% identity). Like NAP-2 and GRO alpha, ENA-78 stimulates neutrophils, inducing chemotaxis, a rise in intracellular free calcium and exocytosis. Cross-desensitization experiments indicate that ENA-78 acts through the same type of receptors as IL-8, NAP-2, and GRO alpha.

Inhibition of interleukin 8 attenuates angiogenesis in bronchogenic carcinoma.

We investigated the role of interleukin 8 (IL-8) in mediating angiogenesis in human bronchogenic carcinoma. Increased quantities of IL-8 were detected in tumor tissue as compared with normal lung tissue. Immunohistochemical staining of tumors revealed primary localization of IL-8 to individual tumor cells and demonstrated the capacity of tumor to elaborate IL-8. Functional studies that used tissue homogenates of tumors demonstrated the induction of both in vitro endothelial cell chemotaxis and in vivo corneal neovascularization. It is important to note that the addition of neutralizing antisera to IL-8 to these assays resulted in the marked and specific attenuation of these responses. Our observations definitively establish IL-8 as a primary mediator of angiogenesis in bronchogenic carcinoma and offer a potential target for immunotherapies against solid malignancies.

Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases.

The success of solid tumor growth and metastasis is dependent upon angiogenesis. Neovascularization within the tumor is regulated, in part, by a dual and opposing system of angiogenic and angiostatic factors. We now report that IP-10, a recently described angiostatic factor, as a potent angiostatic factor that regulates non-small cell lung cancer (NSCLC)-derived angiogenesis, tumor growth, and spontaneous metastasis. We initially found significantly elevated levels of IP-10 in freshly isolated human NSCLC samples of squamous cell carcinoma (SCCA). In contrast, levels of IP-10 were equivalent in either normal lung tissue or adenocarcinoma specimens. The neoplastic cells in specimens of SCCA were the predominant cells that appeared to express IP-10 by immunolocalization. Neutralization of IP-10 in SCCA tumor specimens resulted in enhanced tumor-derived angiogenic activity. Using a model of human NSCLC tumorigenesis in SCID mice, we found that NSCLC tumor growth was inversely correlated with levels of plasma or tumor-associated IP-10. IP-10 in vitro functioned as neither an autocrine growth factor nor as an inhibitor of proliferation of the NSCLC cell lines. Reconstitution of intratumor IP-10 for a period of 8 wk resulted in a significant inhibition of tumor growth, tumor-associated angiogenic activity and neovascularization, and spontaneous lung metastases, whereas, neutralization of IP-10 for 10 wk augmented tumor growth. These findings support the notion that tumor-derived IP-10 is an important endogenous angiostatic factor in NSCLC.

CXCR3 ligands are augmented during the pathogenesis of pulmonary sarcoidosis.

We and other investigators have hypothesised that the CXC chemokine receptor (CXCR)3/CXCR3 ligand biological axis is involved in the formation of sarcoid lung granulomas; however, significant discrepancies in the current literature remain. In an effort to clarify previous conflicting findings, we performed the largest observational study to date of interferon-inducible ELR(-) (lacking the sequence glutamic acid-leucine-arginine) CXC chemokines in sarcoid bronchoalveolar fluid (BALF). BALF chemokine levels from sarcoid patients (n = 72) and healthy controls (n = 8) were measured with the ELISA method. Immunohistochemical staining was performed for CXCR3 and its ligands. BALF CXC chemokine ligand (CXCL)10 levels from sarcoid patients were not significantly increased compared with controls. BALF CXCL11 levels from sarcoid patients demonstrated a trend towards elevation; subgroup analysis by stage showed significant BALF CXCL11 elevation in stage I sarcoid patients compared with controls. BALF CXCL9 levels were elevated from sarcoid patients compared with controls. CXC11, CXCL9 and CXCR3 were expressed from epithelioid histiocytes, multinucleated giant cells and other inflammatory cells forming sarcoid lung granulomas. Our data suggest that CXCL9 and CXCL11 are important mediators in recruiting CXCR3-expressing cells. Importantly, we have made the novel observation that both lymphocytes and cells of monocyte linage express CXCR3 and are involved in the formation of sarcoid lung granulomas.

Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS, and IL-1 beta.

Human endothelial cells produced a neutrophil chemotactic factor (NCF) upon stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or lipopolysaccharide (LPS). The expression of endothelial cell-derived NCF messenger RNA and biological activity was both time- and concentration-dependent. Maximal NCF mRNA expression occurred at 10 and at 2 nanograms per milliliter for TNF and IL-1 beta, respectively; mRNA expression was first observed 1 hour after stimulation and was maintained for at least 24 hours. In situ hybridization analysis showed that NCF mRNA peaked in treated cells by 24 hours, whereas unstimulated cells were negative. These studies demonstrated that endothelial cells may participate in neutrophil-mediated inflammation by synthesizing a chemotactic factor in response to specific monokines and LPS.

Interleukin-8 as a macrophage-derived mediator of angiogenesis.

Angiogenic factors produced by monocytes-macrophages are involved in the pathogenesis of chronic inflammatory disorders characterized by persistent angiogenesis. The possibility was tested that interleukin-8 (IL-8), which is a cytokine that is chemotactic for lymphocytes and neutrophils, is also angiogenic. Human recombinant IL-8 was potently angiogenic when implanted in the rat cornea and induced proliferation and chemotaxis of human umbilical vein endothelial cells. Angiogenic activity present in the conditioned media of inflamed human rheumatoid synovial tissue macrophages or lipopolysaccharide-stimulated blood monocytes was equally blocked by antibodies to either IL-8 or tumor necrosis factor-alpha. An IL-8 antisense oligonucleotide specifically blocked the production of monocyte-induced angiogenic activity. These data suggest a function for macrophage-derived IL-8 in angiogenesis-dependent disorders such as rheumatoid arthritis, tumor growth, and wound repair.

Altered levels of CC chemokines during pulmonary CMV predict BOS and mortality post-lung transplantation.

Pulmonary CMV infection (CMVI) and disease (CMVD) is associated with reduced long-term survival post-lung transplantation, however, the specific biologic mechanisms remain unclear. We have demonstrated a role of CC chemokines during lung allograft dysfunction. Based on these findings, we hypothesized that pulmonary CMV upregulates the expression of multiple CC chemokines that leads to allograft dysfunction and decreased long-term survival. We performed a nested case control study in lung transplant recipients to investigate alterations in CC chemokine biology during pulmonary CMV. Levels of CC chemokines were measured in bronchoalveolar lavage fluid (BALF) from recipients with CMVI (n = 33), CMVD (n = 6), and in healthy lung transplant controls (n = 33). We found a trend toward increased levels of MIP-1alpha/CCL3 during pulmonary CMVI. Levels of MCP-1/CCL2 and RANTES/CCL5 were significantly elevated during pulmonary CMV. Interestingly, elevated levels of CCL3 in BALF were protective with regards to survival. Importantly, elevated levels of CCL2 in BALF predicted the development of BOS, while elevated levels of CCL5 in BALF predicted an increase in mortality post-lung transplant. Altered levels of specific CC chemokines during pulmonary CMV are associated with future clinical outcomes. These results suggest a possible utility of BALF CC chemokines as biomarkers for guiding risk assessment during pulmonary CMV post-lung transplantation.

Anoxia-hyperoxia induces monocyte-derived interleukin-8.

Ischemia-reperfusion and hyperoxia-induced pulmonary injury are associated with the presence of activated neutrophils (PMN) and cellular injury. Although the signals orchestrating the directed migration of these PMN during the pathogenesis of these disease states remain to be fully elucidated, it appears they may be dependent upon the production of certain neutrophil activating/chemotactic factors such as C5a, leukotriene B4, platelet-activating factor, and IL-8. The production of the latter chemotaxin by mononuclear phagocytes is especially intriguing as these cells can mediate inflammatory cell migration by either directly generating IL-8, or by inducing its production from surrounding nonimmune cells. In light of these observations, we propose that ischemia-reperfusion and oxidant stress, in vivo, may be simulated by anoxia-hyperoxia induced stress in vitro, and that this stress may act as a stimulus for the production of IL-8. We now show that isolated human blood monocytes respond to such an oxygen stress with augmented production of IL-8. In initial studies, monocytes demonstrated an increase in the production of IL-8 under anoxic preconditioning. Subsequently, monocytes were cultured under one of the following conditions for 24 h: (a) room air/5% CO2; (b) 95% N2/5% CO2 for 6 h, followed by room air/5% CO2 for 18 h; (c) 95% N2/5% CO2 for 6 h, followed by 95% O2/5% CO2 for 18 h; (d) room air/5% CO2 for 6 h, followed by 95% O2/5% CO2 for 18 h; or (e) 95% O2/5% CO2. Supernatants were isolated and analyzed for IL-8 antigen by specific IL-8 ELISA, demonstrating the production of monocyte-derived IL-8: 5.9 +/- 0.9, 11.4 +/- 1.7, 21.1 +/- 2.3, 14.6 +/- 2.4, and 26.3 +/- 4.7, ng/ml by designated conditions a, b, c, d, and e listed above, respectively. This variance in IL-8 production reflects altered rates of transcription as shown by Northern blot analysis and nuclear run-off assay. Furthermore, when monocytes were concomitantly treated with LPS (100 ng/ml) under in vitro hyperoxic conditions, both IL-8 steady-state mRNA and antigenic activity were two- to threefold greater than under room air conditions. The association of anoxic preconditioning and oxygen stress with augmented production of monocyte-derived IL-8 support the potential role for ischemia-reperfusion and hyperoxia-induced IL-8 production in vivo, providing a possible mechanism for PMN migration/activation in disease states characterized by altered tissue oxygenation.

Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder characterized by inflammation, fibroblast proliferation, and accumulation of extracellular matrix proteins. Leukotrienes (LTs) are pro-inflammatory and pro-fibrogenic mediators derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism. They are thought to play a role in a number of disease processes, but have received relatively little attention in investigations into the pathogenesis of IPF. In this study, we measured the levels of immunoreactive LTs B(4) and C(4) in homogenates of lung tissue obtained from patients with newly diagnosed, untreated IPF, as compared to levels measured in homogenates of uninvolved nonfibrotic lung tissue from patients undergoing resectional surgery for bronchogenic carcinoma. Compared to homogenates on nonfibrotic control lung, homogenates from IPF patients contained 15-fold more LTB(4) and 5-fold more LTC(4). IPF homogenate levels of LTB(4) were significantly correlated with histologic indices of both inflammation (r=0.861) and fibrosis (r=0.926). Activation of 5-LO is known from in vitro studies to be associated with localization of the enzyme at the nuclear membrane. Immunohistochemical staining for 5-LO protein in alveolar macrophages (AMs) demonstrated that such an "activated" localization pattern was significantly more frequent in IPF lung (19.2+/-3.3% of cells) than in control lung (9.3+/-0.9%); this localization pattern was rarely seen (3.2%) in sections from a truly normal transplant donor lung. Consistent with these data, AMs obtained from IPF patients by bronchoalveolar lavage, purified by adherence, and cultured in the absence of a stimulus for 16 h elaborated significantly greater amounts of LTB(4) and LTC(4) than did control AMs obtained from normal volunteers. These data indicate that the 5-LO pathway is constitutively activated in the lungs of patients with IPF, and the AM represents at least one cellular source of LT overproduction in this disorder. We speculate that LTs participate in the pathogenesis of IPF, and their overproduction in this disorder may be amenable to specific pharmacotherapy.

MCP-1 protects mice in lethal endotoxemia.

The overzealous production of proinflammatory cytokines in sepsis can result in shock, multiorgan dysfunction, and even death. In this study, we assessed the role of monocyte chemoattractant protein-1 (MCP-1) as a mediator of sepsis in endotoxin-challenged mice. Intraperitoneal administration of LPS to CD-1 mice induced a substantial time-dependent increase in MCP-1 in plasma, lung, and liver. The passive immunization of mice with rabbit antimurine MCP-1 antiserum 2 h before endotoxin administration resulted in a striking increase in LPS-induced mortality from 10% in control animals to 65% in anti-MCP-1-treated animals. Importantly, the administration of anti-MCP-1 antibodies to endotoxin-challenged mice resulted in increases in peak TNF-alpha and IL-12 levels, and also in a trend toward decreased serum levels of IL-10. Conversely, the administration of recombinant murine MCP-1 intraperitoneally significantly protected mice from endotoxin-induced lethality, and resulted in an increase in IL-10 levels, a decrease in IL-12 levels, and a trend toward decreased levels of TNF. In conclusion, our findings indicate that MCP-1 is a protective cytokine expressed in murine endotoxemia, and does so by shifting the balance in favor of antiinflammatory cytokine expression in endotoxin-challenged animals.

Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice.

The salient feature of solid tumor growth is the strict dependence on local angiogenesis. We have previously demonstrated that IL-8 is an angiogenic factor present in freshly isolated specimens of human non-small cell lung cancer (NSCLC). Using a model of human NSCLC tumorigenesis in SCID mice, we now report that IL-8 acts as a promoter of human NSCLC tumor growth through its angiogenic properties. Passive immunization with neutralizing antibodies to IL-8 resulted in more than 40% reduction in tumor size and was associated with a decline in tumor-associated vascular density and angiogenic activity. IL-8 did not act as an autocrine growth factor for NSCLC proliferation. The reduction in primary tumor size in response to neutralizing antibodies to IL-8 was also accompanied by a trend toward a decrease in spontaneous metastasis to the lung. These data support the notion that IL-8 plays a significant role in mediating angiogenic activity during tumorigenesis of human NSCLC, thereby offering a potential target for immunotherapy against solid tumors.

Role of tumor necrosis factor-alpha in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat.

Cytokines are recognized as critical early mediators of organ injury. We attempted to determine whether or not severe hepatic ischemia/reperfusion injury results in tumor necrosis factor-alpha (TNF-alpha) release with subsequent local and systemic tissue injury. After 90 min of lobar hepatic ischemia, TNF was measurable during the reperfusion period in the plasma of all 14 experimental animals, with levels peaking between 9 and 352 pg/ml. Endotoxin was undetectable in the plasma of these animals. Pulmonary injury, as evidenced by a neutrophilic infiltrate, edema and intra-alveolar hemorrhage developed after hepatic reperfusion. The neutrophilic infiltrate was quantitated using a myeloperoxidase (MPO) assay; this demonstrated a significant increase in MPO after only 1 h of reperfusion. Anti-TNF antiserum pretreatment significantly reduced the pulmonary MPO after hepatic reperfusion. After a 12-h reperfusion period, there was histologic evidence of intra-alveolar hemorrhage and pulmonary edema. Morphometric assessment showed that pretreatment with anti-TNF antiserum was able to completely inhibit the development of pulmonary edema. Liver injury was quantitated by measuring serum glutamic pyruvic transaminase which showed peaks at 3 and 24 h. Anti-TNF antiserum pretreatment was able to significantly reduce both of these peak elevations. These data show that hepatic ischemia/reperfusion results in TNF production, and that this TNF is intimately associated with pulmonary and hepatic injury.

Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44.

Hyaluronan (HA) is a glycosaminoglycan constituent of extracellular matrix. In its native form HA exists as a high molecular weight polymer, but during inflammation lower molecular weight fragments accumulate. We have identified a collection of inflammatory genes induced in macrophages by HA fragments but not by high molecular weight HA. These include several members of the chemokine gene family: macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, cytokine responsive gene-2, monocyte chemoattractant protein-1, and regulated on activation, normal T cell expressed and secreted. HA fragments as small as hexamers are capable of inducing expression of these genes in a mouse alveolar macrophage cell line, and monoclonal antibody to the HA receptor CD44 completely blocks binding of fluorescein-labeled HA to these cells and significantly inhibits HA-induced gene expression. We also investigated the ability of HA fragments to induce chemokine gene expression in human alveolar macrophages from patients with idiopathic pulmonary fibrosis and found that interleukin-8 mRNA is markedly induced. These data support the hypothesis that HA fragments generated during inflammation induce the expression of macrophage genes which are important in the development and maintenance of the inflammatory response.

MIP-1alpha as a critical macrophage chemoattractant in murine wound repair.

At sites of injury, macrophages secrete growth factors and proteins that promote tissue repair. While this central role of the macrophage has been well studied, the specific stimuli that recruit macrophages into sites of injury are not well understood. This study examines the role of macrophage inflammatory protein 1alpha (MIP-1alpha), a C-C chemokine with monocyte chemoattractant capability, in excisional wound repair. Both MIP-1alpha mRNA and protein were detectable in murine wounds from 12 h through 5 d after injury. MIP-1alpha protein levels peaked 3 d after injury, coinciding with maximum macrophage infiltration. The contribution of MIP-1alpha to monocyte recruitment into wounds was assessed by treating mice with neutralizing anti-MIP-1alpha antiserum before injury. Wounds of mice treated with anti-MIP-1alpha antiserum had significantly fewer macrophages than control (41% decrease, P < 0. 01). This decrease in wound macrophages was paralleled by decreased angiogenic activity and collagen synthesis. When tested in the corneal micropocket assay, wound homogenates from mice treated with anti-MIP-1alpha contained significantly less angiogenic activity than control wound homogenates (27% positive for angiogenic activity versus 91% positive in the control group, P < 0.01). Collagen production was also significantly reduced in the wounds from anti-MIP-1alpha treated animals (29% decrease, P < 0.05). The results demonstrate that MIP-1alpha plays a critical role in macrophage recruitment into wounds, and suggest that appropriate tissue repair is dependent upon this recruitment.

Polyclonal antibody directed against human RANTES ameliorates disease in the Lewis rat adjuvant-induced arthritis model.

Adjuvant-induced arthritis (AIA) is one of many animal models of rheumatoid arthritis, a disease characterized by a T-lymphocyte and macrophage cellular infiltrate. We have characterized the development of this disease model with respect to chemokine expression. Increased levels of two chemokines, RANTES, a T-lymphocyte and monocyte chemo-attractant, and KC a chemoattractant for neutrophils, were found in whole blood and in the joint. Surprisingly, levels of MIP-1alpha, another T-lymphocyte and monocyte chemoattractant, were unchanged throughout the course of the disease in whole blood and only slightly elevated in the joint. RANTES expression plays an important role in the disease since a polyclonal antibody to RANTES greatly ameliorated symptoms in animals induced for AIA and was found to be as efficacious as treatment with indomethacin, a non-steroidal anti inflammatory. Polyclonal antibodies to either MIP-1alpha or KC were ineffective. This is the first report to show the importance of RANTES in the development of AIA.

Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin E2 and to express cyclooxygenase-2.

Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen synthesis. In this study, we compared lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (F-IPF) and from patients undergoing resectional surgery for lung cancer (F-nl) with respect to their capacity for PGE2 synthesis and their expression and regulation of cyclooxygenase (COX) proteins. Basal COX activity, assessed by quantitating immunoreactive PGE2 synthesized from arachidonic acid, was twofold less (P < 0.05) in F-IPF than F-nl. In F-nl, incubation with the agonists PMA, LPS, or IL-1 increased COX activity and protein expression of the inducible form of COX, COX-2, and these responses were inhibited by coincubation with dexamethasone. By contrast, F-IPF failed to demonstrate increases in COX-2 protein expression or COX activity in response to these agonists. Under conditions of maximal induction, COX activity in F-IPF was sixfold less than that in F-nl (P < 0.05). Our data indicate that F-IPF have a striking defect in their capacity to synthesize the antiinflammatory and antifibrogenic molecule PGE2, apparently because of a diminished induction of COX-2 protein. This reduction in the endogenous capacity of F-IPF to down-regulate their function via PGE2 may contribute to the inflammatory and fibrogenic response in IPF. Moreover, we believe that this represents the first description of a defect in COX-2 expression in association with a human disease.

Interleukin-10 expression and chemokine regulation during the evolution of murine type II collagen-induced arthritis.

In the enclosed study we have examined the expression and contribution of specific chemokines, macrophage inflammatory protein 1 alpha (MIP-1 alpha) and macrophage inflammatory protein 2 (MIP-2), and interleukin 10 (IL-10) during the evolution of type II collagen-induced arthritis (CIA). Detectable levels of chemotactic cytokine protein for MIP-1 alpha and MIP-2 were first observed between days 32 and 36, after initial type II collagen challenge, while increases in IL-10 were found between days 36 and 44. CIA mice passively immunized with antibodies directed against either MIP-1 alpha or MIP-2 demonstrated a delay in the onset of arthritis and a reduction of the severity of arthritis. On the contrary, CIA mice receiving neutralizing anti-IL-10 antibodies demonstrated an acceleration of the onset and an increase in the severity of arthritis. Interestingly, anti-IL-10 treatment increased the expression of MIP-1 alpha and MIP-2, as well as increased myeloperoxidase (MPO) activity and leukocyte infiltration in the inflamed joints. These data suggest that MIP-1 alpha and MIP-2 play a crucial role in the initiation and maintenance, while IL-10 appears to play a regulatory role during the development of experimental arthritis.