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Angiogenesis, the growth of new blood vessels, is vital to the ingress of inflammatory leukocytes in rheumatoid arthritis (RA) synovial tissue and to the growth and proliferation of RA pannus. The factors that mediate the growth of new blood vessels have not been completely defined. This study examined the ability of Glu-Leu-Arg (ELR)-containing chemokines to induce angiogenesis in the RA joint.To reflect angiogenic activity in vivo, we selected a model using whole human synovial tissue rather than isolated cells. Tissues were examined by immunohistochemistry and enzyme-linked immunosorbent assay, and tissue homogenates were immunoneutralized and assayed for their ability to induce endothelial cell chemotaxis and rat corneal neovascularization.Cells expressing interleukin-8 (IL-8) and epithelial neutrophil activating peptide 78 (ENA-78) were located in proximity to factor VIII-related antigen-immunopositive endothelial cells. RA homogenates produced more IL-8 and ENA-78 compared with normal synovial tissue homogenates. Moreover, homogenates from RA synovial tissue produced significantly more chemotactic activity for endothelial cells in vitro and angiogenic activity in the rat cornea in vivo than did normal synovial tissue homogenates. The effects of IL-8 and ENA-78 accounted for a significant proportion of the chemotactic activity of endothelial cells and angiogenic activity found in RA synovial tissue homogenates.These results indicate that the ELR-containing chemokines IL-8 and ENA-78 are important contributors to the angiogenic activity found in the inflamed RA joint. It is possible that efforts aimed at down-regulating these chemokines offer a novel targeted therapy for the treatment of RA.

Paracetamol-induced hepatic necrosis is the most common form of toxic liver injury experienced in clinical practice in the UK and USA. Recently, reports have described prevention of hepatic necrosis, induced by other hepato-toxins, by inhibiting tumour necrosis factor alpha (TNFalpha). The aim of the present study was to determine the role of TNFalpha in paracetamol-induced hepatic necrosis. Six-week-old CBA/J female mice were given 300 mg/kg paracetamol by intraperitoneal (IP) injection after an 8-h fast. Hepatic expression of TNFalpha was measured by enzyme-linked immunoassay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Serum TNFalpha was measured by ELISA. One hour prior to paracetamol injection, mice were also given blocking anti-TNFalpha antibodies, soluble TNFalpha receptor, interleukin 10 (IL-10), and dexamethasone. Hepatic injury was measured by serum aspartate aminotransferase and histological assessment on haematoxylin and eosin (HE)-stained liver sections. There was a significant increase in serum TNFalpha at 6 h (control 0.002+/-0.002 ng/ml, n=7; paracetamol-treated 0.022+/-0.007 ng/ml, n=5, p0.05), but hepatic TNFalpha expression did not change up to 24 h following paracetamol injection. Histologically severe centrilobular hepatic necrosis was noted at 3 h and progressed for 24 h after paracetamol poisoning. Death rate, serum aspartate aminotransferase, and hepatic histology were not significantly different between the groups treated with blocking anti-TNFalpha antibodies, soluble TNFalpha receptor, IL-10, and dexamethasone, compared with controls. In conclusion, there is no evidence to suggest that modulation of TNFalpha expression affects hepatic injury following experimental paracetamol poisoning; anti-TNFalpha therapies are therefore unlikely to be effective in the corresponding clinical situation.

Invasive pulmonary aspergillosis is a devastating complication of immunosuppression, which occurs in association with neutrophil dysfunction or deficiency. ELR+ CXC chemokines are a subfamily of chemokines that play a critical role in neutrophil chemotaxis and activation both in vitro and in vivo. We hypothesized that interaction of these ligands with CXC chemokine receptor-2 (CXCR2), their sole murine receptor, is a major component of neutrophil-dependent pulmonary host defense against Aspergillus fumigatus. In immunocompetent animals, neutrophils were recruited to the lung in response to intratracheally administered A. fumigatus conidia. In a model of transient in vivo depletion of neutrophils, animals developed invasive pulmonary aspergillosis, associated with delayed influx of neutrophils into the lung. In both normal and neutrophil-depleted animals, the ELR+ CXC chemokines MIP-2 and KC were induced in response to intratracheal administration of conidia. Ab-mediated neutralization of the common ELR+ CXC chemokine receptor, CXCR2, resulted in development of invasive disease indistinguishable from the disease in neutrophil-depleted animals, while control animals were highly resistant to the development of infection. CXCR2 neutralization was associated with reduced lung neutrophil influx and resulted in a marked increase in mortality compared with controls. In contrast, animals with constitutive lung-specific transgenic expression of KC were resistant to the organism, with reduced mortality and lower lung burden of fungus. We conclude that CXCR2 ligands are essential mediators of host defense against A. fumigatus, and may be important targets in devising future therapeutic strategies in this disease.

Few studies have addressed the importance of vascular remodeling in the lung during the development of bleomycin-induced pulmonary fibrosis (BPF). For fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during BPF are dependent in part on a relative deficiency of the angiostatic CXC chemokine, IFN-gamma-inducible protein-10 (IP-10). To test this hypothesis, we measured IP-10 by specific ELISA in whole lung homogenates in either bleomycin-treated or control mice and correlated these levels with lung hydroxyproline. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a decrease in the presence of IP-10 that was correlated to a greater angiogenic response and total lung hydroxyproline content. Systemic administration of IP-10 significantly reduced BPF without any alteration in lung lymphocyte or NK cell populations. This was also paralleled by a reduction in angiogenesis. Furthermore, IP-10 had no direct effect on isolated pulmonary fibroblasts. These results demonstrate that the angiostatic CXC chemokine, IP-10, inhibits fibroplasia and deposition of extracellular matrix by regulating angiogenesis.

Recent studies suggest that monocyte chemoattractant protein-1 (MCP-1) is involved in fibrosis through the regulation of profibrotic cytokine generation and matrix deposition. Changes in MCP-1, C-C chemokine receptor 2 (CCR2), procollagen I and III, and TGF beta were examined in fibroblasts cultured from normal lung and from nonfibrotic (i.e., Th1-type) and fibrotic (i.e., Th2-type) pulmonary granulomas. Th2-type fibroblasts generated 2-fold more MCP-1 than similar numbers of Th1-type or normal fibroblasts after 24 h in culture. Unlike normal and Th1-type fibroblasts, Th2-type fibroblasts displayed CCR2 mRNA at 24 h after IL-4 treatment. By flow cytometry, CCR2 was present on 40% of untreated Th2-type fibroblasts, whereas CCR2 was present on20% of normal and Th1-type fibroblasts after similar treatment. IL-4 increased the number of normal fibroblasts with cell-surface CCR2 but IFN-gamma-treatment of normal and Th2-type fibroblasts significantly decreased the numbers of CCR2-positive cells in both populations. Western blot analysis showed that total CCR2 protein expression was markedly increased in untreated Th2-type fibroblasts compared with normal and Th1-type fibroblasts. IL-4 treatment enhanced CCR2 protein in Th1- and Th2-type fibroblasts whereas IFN-gamma treatment augmented CCR2 protein in normal and Th1-type fibroblasts. All three fibroblast populations exhibited MCP-1-dependent TGF-beta synthesis, but only normal and Th2-type fibroblasts showed a MCP-1 requirement for procollagen mRNA expression. Taken together, these findings suggest that lung fibroblasts are altered in their expression of MCP-1, TGF-beta, CCR2, and procollagen following their participation in pulmonary inflammatory processes, and these changes may be important during fibrosis.

Bronchial eosinophil and mononuclear cell infiltrates are a hallmark of the asthmatic lung and are associated with the induction of reversible airway hyperreactivity. In these studies, we have found that monocyte chemotactic protein-1 (MCP-1), a CC (beta) chemokine, mediates airway hyperreactivity in normal and allergic mice. Using a murine model of cockroach Ag-induced allergic airway inflammation, we have demonstrated that anti-MCP-1 Abs inhibit changes in airway resistance and attenuate histamine release into the bronchoalveolar lavage, suggesting a role for MCP-1 in mast cell degranulation. In normal mice, instillation of MCP-1 induced prolonged airway hyperreactivity and histamine release. In addition, MCP-1 directly induced pulmonary mast cell degranulation in vitro. These latter effects would appear to be selective because no changes were observed when macrophage-inflammatory protein-1alpha, eotaxin, or MCP-3 were instilled into the airways of normal mice or when mast cells were treated in vitro. Airway hyperreactivity was mediated by MCP-1 through CCR2 because allergen-induced as well as direct MCP-1 instilled-induced changes in airway hyperreactivity were significantly attenuated in CCR2 -/- mice. The neutralization of MCP-1 in allergic animals and instillation of MCP-1 in normal animals was related to leukotriene C4 levels in the bronchoalveolar lavage and was directly induced in pulmonary mast cells by MCP-1. Thus, these data identify MCP-1 and CCR2 as potentially important therapeutic targets for the treatment of hyperreactive airway disease.

The chemokine, epithelial neutrophil-activating peptide-78 (ENA-78), is a potent neutrophil chemotaxin whose expression is increased in inflamed synovial tissue and fluid in human rheumatoid arthritis compared with osteoarthritis. Since ENA-78 has been implicated in the pathogenesis of RA, we examined the expression of an ENA-78-like protein during the development of rat adjuvant-induced arthritis (AIA). Using an ELISA assay, we found increased levels of antigenic ENA-78-like protein in the sera of AIA animals compared with control normal animals by day 7 postadjuvant injection. ENA-78-like protein levels continued to increase as AIA developed. ENA-78-like protein levels in joint homogenates were increased in AIA animals later in the development of the disease, by day 18 during maximal arthritis, compared with control animals. Expression of ENA-78-like protein in both the AIA serum and joint correlated with the progression of inflammation of the joints. Anti-human ENA-78 administered before disease onset modified the severity of AIA, while administration of anti-ENA-78 after clinical onset of AIA did not modify the disease. These data support a role for an ENA-78-like protein as an important chemokine in the progression and maintenance of AIA.

Invasive pulmonary aspergillosis is a common and devastating complication of immunosuppression, whose incidence has increased dramatically in tandem with the increase in the number of immunocompromised patients. Given the role of TNF-alpha in other pulmonary infections, we hypothesized that TNF-alpha is an important proximal signal in murine invasive pulmonary aspergillosis. Intratracheal challenge with Aspergillus fumigatus conidia in both neutropenic (cyclophosphamide-treated) and nonneutropenic BALB/c mice resulted in the time-dependent increase in lung TNF-alpha levels, which correlated with the histologic development of a patchy, peribronchial infiltration of mononuclear and polymorphonuclear cells. Ab-mediated neutralization of TNF-alpha resulted in an increase in mortality in both normal and cyclophosphamide-treated animals, which was associated with increased lung fungal burden as determined by histology and as quantified by chitin content. Depletion of TNF-alpha resulted in a reduced lung neutrophil influx in both normal and cyclophosphamide-treated animals, which occurred in association with a decrease in lung levels of the C-X-C chemokine, macrophage inflammatory protein-2 and the C-C chemokines macrophage inflammatory protein-1alpha and JE. In cyclophosphamide-treated animals, intratracheal administration of a TNF-alpha agonist peptide (TNF70-80) 3 days before, but not concomitant with, the administration of Aspergillus conidia resulted in improved survival from 9% in control mice to 55% in TNF70-80-treated animals. These studies indicate that TNF-alpha is a critical component of innate immunity in both immunocompromised and immunocompetent hosts, and that pretreatment with a TNF-alpha agonist peptide in a compartmentalized fashion can significantly enhance resistance to A. fumigatus in neutropenic animals.

To explore the mechanism of immunosuppression associated with sepsis, we developed a murine model of sepsis-induced Pseudomonas aeruginosa pneumonia. CD-1 mice underwent either cecal ligation and 26-gauge needle puncture (CLP) or sham surgery, followed by the intratracheal (i.t.) administration of P. aeruginosa or saline. Survival in mice undergoing CLP followed 24 h later by the i.t. administration of saline or P. aeruginosa was 58% and 10%, respectively, whereas 95% of animals undergoing sham surgery followed by P. aeruginosa administration survived. Increased mortality in the CLP/P. aeruginosa group was attributable to markedly impaired lung bacterial clearance and the early development of P. aeruginosa bacteremia. The i.t. administration of bacteria to CLP-, but not sham-, operated mice resulted in an impressive intrapulmonary accumulation of neutrophils. Furthermore, P. aeruginosa challenge in septic mice resulted in a relative shift toward enhanced lung IL-10 production concomitant with a trend toward decreased IL-12. The i.p., but not i.t., administration of IL-10 Abs given just before P. aeruginosa challenge in septic mice significantly improved both survival and clearance of bacteria from the lungs of septic animals administered P. aeruginosa. Finally, alveolar macrophages isolated from animals undergoing CLP displayed a marked impairment in the ability to ingest and kill P. aeruginosa ex vivo, and this defect was partially reversed by the in vivo neutralization of IL-10. Collectively, these observations indicate that the septic response substantially impairs lung innate immunity to P. aeruginosa, and this effect is mediated primarily by endogenously produced IL-10.

The increase in inner-city asthma among children appears to be due to allergic responses to several allergens. Recent studies have demonstrated that Ags derived from cockroaches are especially prominent in these settings and a significant health concern for the induction of asthma in children. In the present study, we have outlined the development of a murine model of cockroach allergen-induced airway disease and assessed specific mechanisms of the response, which resembles atopic human asthma. The allergic responses in this model include allergen-specific airway eosinophilia and significantly altered airway physiology, which directly correlates with inflammation. We have further utilized this allergen to establish primary and secondary rechallenge stages of late phase hyperreactivity exacerbation. This latter stage is characterized by greater changes in airway physiology than the primary stage, and it is likely due to the preexisting peribronchial inflammation present at the time of the second allergen rechallenge. We have identified specific roles for CC chemokines during these stages, with MIP-1alpha being an important eosinophil attractant during the primary stage and eotaxin during the secondary rechallenge stage. The development of these models allows the evaluation of mediators involved in both stages of cockroach allergen challenge, as well as the testing of specific therapeutic modalities.

Eotaxin participation was analyzed during types 1 and 2 lung granuloma formation induced by embolizing Sepharose beads coupled to purified protein derivative (PPD) of Mycobacterium bovis or soluble Ags derived from Schistosoma mansoni eggs. Eotaxin was monitored by protein ELISA and semiquantitative reverse-transcriptase PCR mRNA analysis. Both types 1 and 2 granulomas released eotaxin, but levels were sixfold greater (on day 4) in the type 2 than for the type 1 or foreign body granulomas. Transcripts for eotaxin, IL-4, and CCR3 (eotaxin receptor) were also enhanced during type 2 granuloma formation. Anti-IL-4 treatment impaired eotaxin mRNA in lungs with type 2 granulomas, indicating that IL-4 promoted local eotaxin expression. In vivo, anti-eotaxin treatment caused modest reductions in the size of both types 1 and 2 lesions, with negligible effect on eosinophil recruitment. Surprisingly, anti-eotaxin treatment abrogated IFN-gamma-producing cells in regional lymph nodes during the type 1 PPD response. Lymph nodes draining both types 1 and 2 lesions showed enhanced CCR3 mRNA, but this followed the time of maximum eotaxin protein and mRNA expression. Correlative, in vitro studies revealed that graded doses of eotaxin increased IFN-gamma production from PPD-sensitive regional lymph node cultures, while monocyte-chemotactic protein-1, an important macrophage chemoattractant, had the opposite effect. These findings indicate that eotaxin expression is not limited to type 2 hypersensitivity granulomas, but also promotes IFN-gamma production during mycobacterial responses.

A vigorous host response is required to effectively clear pathogenic bacteria from the lungs and is dependent upon the recruitment and activation of neutrophils and macrophages. A family of chemotactic cytokines, referred to as chemokines, have been shown to participate in this complex protective response. In this study, we assessed the role of the C-X-C chemokine KC in lung antibacterial host defense using wild-type (wt) B6D2 mice or transgenic mice that had been bred on a B6D2 background expressing KC under the control of a Clara cell-specific promoter within the lung. The administration of Klebsiella pneumoniae to both wt and KC-transgenic mice resulted in a time-dependent expression of KC protein within the lung that peaked at 24 to 48 h postinoculation. When infected with K. pneumoniae, the KC-transgenic mice showed a striking improvement in survival compared with wt control mice. This improved survival was due to an increase in bacterial clearance, which occurred in association with a vigorous recruitment of neutrophils in the KC-transgenic mice compared with their wt control counterparts. No differences in the lung levels of the specific cytokines TNF-alpha, IFN-gamma, IL-12, and IL-10 were noted. However, inducible macrophage inflammatory protein-2 levels were significantly decreased in the KC-transgenic mice compared with the wt mice. This study indicates that the compartmentalized overexpression of KC in vivo results in increased lung bacterial clearance and improved survival, which occurs in association with enhanced polymorphonuclear leukocyte influx to the lung.

This study addressed the role of endogenous monocyte chemoattractant protein-1 (MCP-1) in Ag-stimulated lymphokine synthesis and proliferation by CD4+ T cells during their coculture with purified lung fibroblasts or splenic macrophages. Initial experiments showed that fibroblasts exposed to IL-4, TNF alpha, or IL-4 and TNF-alpha (all at 10 ng/ml) for 24 h released five- to eightfold more MCP-1 than similarly treated splenic macrophages. In 72-h coculture experiments, the synthesis of IL-4 by OVA-activated CD4+ T cells added to lung fibroblasts or splenic macrophages was significantly inhibited when endogenous MCP-1 was neutralized using polyclonal anti-MCP-1 antiserum. In these same cocultures, IFN-gamma levels were significantly enhanced. Similarly, IFN-gamma levels were significantly enhanced in 72-h cocultures of a purified peptide derivative-activated CD4+ Th1 clone and lung fibroblasts or splenic macrophages following immunoneutralization of MCP-1. In separate experiments, the selective inhibition of MCP-1 synthesis by lung fibroblasts and splenic macrophages using an MCP-1 antisense oligonucleotide significantly enhanced the proliferation of CD4+ T cells during a 96-h coculture. Taken together, these data suggest that MCP-1 exerts an immunomodulatory effect on CD4+ T cell-derived IL-4 and IFN-gamma release and CD4+ T cell proliferation during cell-to-cell interactions.

Pulmonary granulomatous inflammation modulated by IFN-gamma and IL-12 is also associated with augmented inducible nitric oxide synthase (NOS II). To address the role of increased nitric oxide synthesis in this model, mice received daily i.p. injections of NG-nitro-L-arginine-methyl ester (L-NAME; 8 mg/kg) during both the 2-wk immunization period with purified protein-derivative (PPD) and the subsequent lung challenge with PPD-coated Sepharose beads. Other groups of animals received saline, L-NAME or NG-nitro-D-arginine-methyl ester (D-NAME; 8 mg/kg) during the pulmonary embolization period and not the PPD sensitization period. On day 4 post-PPD bead challenge, PCR analysis of the whole lung revealed that NOS II expression appeared to be similar in both of the L-NAME treatment protocols. L-NAME-treated mice in both dosing protocols had lung lesions that were significantly larger than granuloma lesions measured in mice that received saline or D-NAME. The enlarged lesions from L-NAME-treated mice contained markedly greater numbers of neutrophils and eosinophils. Equivalent numbers of PPD-activated dispersed cells from whole lungs of L-NAME-treated mice produced significantly higher levels of IL-4 and IL-10 and smaller amounts of IL-12 and IFN-gamma compared with similar lung cultures derived from control or D-NAME-treated mice. Levels of C-C chemokines such as monocyte chemoattractant protein-1 (MCP-1), C10, and macrophage inflammatory protein-1alpha (MIP-1alpha) were also significantly elevated in lung cultures from L-NAME-treated mice compared with controls. Thus, nitric oxide regulates the size and cellular composition of the Th1-type lung granuloma, possibly through its effects on the cytokine and chemokine profile associated with this lesion.

Turnover of the extracellular matrix (ECM), activation of macrophages, and accumulation of chemokines/cytokines are all hallmarks of chronic inflammation. Extracellular matrix components, such as hyaluronan (HA), have recently been shown to influence macrophage effector functions, such as the release of inflammatory chemokines and cytokines. Although low m.w. fragments of the glycosaminoglycan HA induce macrophages to secrete numerous inflammatory mediators, the mechanisms regulating ECM-induced macrophage activation are poorly understood. We have examined the effects of IL-10 and IFN-gamma on HA-induced chemokine gene expression in primary mouse macrophages. We found that IL-10 and IFN-gamma independently inhibit HA-induced expression of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and KC at both the mRNA and protein levels. Whereas IL-10 inhibited most of the HA-induced chemokines tested, IFN-gamma selectively inhibited only MIP-1alpha, MIP-1beta, and KC. This inhibition did not require prestimulation and occurred even when the cytokines were added up to 3 h after stimulation with HA. For MIP-1alpha, the inhibition by IFN-gamma occurred at the level of transcription, whereas IL-10 predominantly decreased the stability of MIP-1alpha mRNA. IFN-gamma and IL-10 equally inhibited macrophage expression of MIP-1beta mRNA at the level of transcription, but MIP-1beta mRNA stability was decreased to a greater extent by IL-10. These data identify a previously unrecognized role for IL-10 and IFN-gamma as regulators of ECM-induced macrophage expression of inflammatory chemokines.

Beginning at the time of insulitis, nonobese diabetic (NOD) mice demonstrate a thymocyte and peripheral T cell proliferative hyporesponsiveness induced by TCR cross-linking, which is associated with reduced IL-2 and IL-4 secretion. We previously reported that NOD CD4+ T cell hyporesponsiveness is reversed completely in vitro by exogenous IL-4, and that administration of IL-4 to NOD mice prevents the onset of insulin-dependent diabetes mellitus (IDDM). This result suggested that T cell-mediated destruction of pancreatic islet beta cells may result from a hyporesponsiveness in regulatory Th2 cells favoring a Th1 cell-mediated environment in the pancreas. In the present study, we tested this possibility by analysis of the mechanisms of protection from IDDM afforded by IL-4 treatment in NOD mice. We show that IL-4 protects NOD mice from insulitis and IDDM when administered i.p. three times a week for 10 wk beginning at 2 wk of age. This occurs by the modulation of the homing of autoreactive cells to inflammatory sites and the stabilization of a protective Th2-mediated environment in the thymus, spleen, and pancreatic islets. Thus, IL-4 treatment favors the expansion of regulatory CD4+ Th2 cells in vivo and prevents the onset of insulitis and IDDM mediated by autoreactive Th1 cells.

Components of the extracellular matrix (ECM) can regulate leukocyte activation and function at inflammatory sites. Low molecular weight fragments of the ECM glycosaminoglycan hyaluronan (LMW-HA) that accumulate in inflammation, but not the ubiquitous high molecular weight form of HA (HMW-HA), have been shown to induce cytokine and/or chemokine production by alveolar and bone-marrow derived macrophages. To determine the cellular requirements for responsiveness to HA, we compared the effects of HMW-HA and LMW-HA on resident and thioglycollate-elicited murine peritoneal macrophages. We demonstrate that treatment of elicited macrophages with LMW-HA, but not with HMW-HA, stimulated production of the chemokines RANTES and macrophage inflammatory protein-1alpha and -1beta. Further, we demonstrate that LMW-HA induced the production of biologically active IL-12, a proinflammatory cytokine not previously known to be regulated by cell-matrix interactions. The LMW-HA-induced production of IL-12 by elicited macrophages was inhibited by an anti-CD44 mAb that blocks HA binding. In contrast to elicited macrophages, freshly explanted resident peritoneal macrophages did not respond to LMW-HA. However, preculture in vitro before stimulation led to adhesion-dependent priming for LMW-HA-induced cytokine and chemokine production by resident macrophages. These results provide further evidence of the potential importance of CD44/LMW-HA interactions in regulating the immune response at sites of inflammation and demonstrate that the state of differentiation of macrophages may determine their sensitivities to matrix components.

Our objective was to define the role of the chemokine macrophage-inflammatory protein-1alpha (MIP-1alpha) in the efferent phase of pulmonary cell-mediated immunity (CMI) against Cryptococcus neoformans. Following intratracheal inoculation of C. neoformans (24067) into CBA/J mice, the development of CMI was required for leukocyte recruitment into the lungs at 2 wk postinfection. MIP-1alpha mRNA was expressed by day 6 postinfection, and MIP-1alpha protein in bronchoalveolar lavage fluid was detectable at day 6, but significantly elevated at days 19 and 33. Administration of neutralizing anti-MIP-1alpha Abs from days 7 to 13 blocked the increase in bronchoalveolar lavage fluid MIP-1alpha and resulted in a 37% decrease in total leukocytes in the lungs at day 16. There were 66% fewer macrophages/monocytes and 42% fewer neutrophils in the lungs of anti-MIP-1alpha-treated mice, and the pulmonary burden of C. neoformans was threefold higher. There was no significant difference in the number of eosinophils, CD4+, CD8+, or B220+ lymphocytes between the two groups of mice. Neutralization of MIP-1alpha did not significantly decrease the levels of monocyte chemotactic protein-1 (MCP-1); however, neutralization of MCP-1 significantly decreased MIP-1alpha levels, demonstrating that induction of MIP-1alpha was largely dependent on MCP-1 production. Neutralization of MIP-1alpha also blocked the cellular recruitment phase of a recall response to cryptococcal Ag in the lungs of immunized mice. Thus, in both the contexts of active cryptococcal infection or rechallenge with cryptococcal Ag, MIP-1alpha was required during the efferent phase of CMI for maximal leukocyte recruitment into the lungs, most notably the recruitment of phagocytic effector cells (neutrophils and macrophages).

The development of T cell immunity is required to clear a pulmonary Cryptococcus neoformans infection (via the recruitment and activation of inflammatory cells). The objective of our studies was to determine whether TNF-alpha is required during the afferent phase of the response. The levels of TNF-alpha mRNA and protein in the lungs increased following intratracheal inoculation of CBA/J mice with C. neoformans 52 and preceded the development of an inflammatory response in the lungs. Administration of neutralizing TNF-alpha-specific antiserum on days 0, 3, 6, and 9 reduced inflammatory cell recruitment by 80% on day 13, resulting in a 3-fold increase in lung C. neoformans CFU. The number of CD4+ T cells was decreased by 65%, the number of neutrophils was decreased by 84%, and the number of macrophages was decreased by98%. Strikingly, a single dose of neutralizing anti-TNF-alpha serum was sufficient to prevent the development of protective cell-mediated immunity on day 35 if administered on day 0, but was ineffective if delayed until day 14. Day 0 anti-TNF-alpha-treated mice could not generate cryptococcus-specific delayed type hypersensitivity reactivity, clear the infection from their lungs (10(4)-fold increase in CFU), control dissemination to the spleen and brain (10(5)- and 10(6)-fold increases in CFU), or adoptively transfer cryptococcus-specific immunity (mononuclear cell recruitment into the lungs following intratracheal challenge or footpad delayed type hypersensitivity). Thus, the production of TNF-alpha during the afferent phase of the immune response against a pulmonary C. neoformans infection (before day 14) is critical for the development of protective T cell immunity in both the lungs and extrapulmonary sites.

Obliterative bronchiolitis (OB) is a devastating complication in lung transplantation. We postulated that the pathogenesis of OB is mediated, in part, by neutrophils. We serially collected bronchoalveolar lavage (BAL) fluid from lung transplant recipients. Patients were divided into two groups depending on the presence or absence of OB. Samples from patients who never developed OB were further divided according to whether rejection was present. These samples were labeled healthy or rejection. Samples from patients who developed OB were divided according to whether the sample was obtained before (future OB) or at the time of diagnosis of OB (OB). The OB group, as compared with the healthy and rejection group, had significantly elevated neutrophil counts (3.9 x 10(5) +/- 1.8 x 10(5) vs 0.3 x 10(5) +/- 0.07 x 10(5) and 0.4 x 10(5) +/- 0.1 x 10(5), respectively, p0.01 for both) and levels of IL-8 (3131 +/- 1468 pg/ml vs 240 +/- 62 pg/ml and 172 +/- 47 pg/ml, p0.01 for both). Furthermore, we demonstrated immunolocalization of IL-8 associated with alpha smooth muscle actin-positive cells in the peribronchial region of OB. To confirm that the IL-8 present in BAL fluid from patients with OB was bioactive, we performed neutrophil chemotaxis experiments that showed that IL-8 accounted for a significant amount of the neutrophil chemotactic activity. We also found a trend toward higher levels of neutrophils and IL-8 in BALs from the future OB as compared with the healthy group (7.1 x 10(4) +/- 4.2 x 10(4) vs 3.4 x 10(4) +/- 0.7 x 10(4) and 500 +/- 306 pg/ml vs 240 +/- 62 pg/ml). In conclusion, we have provided the novel observation that in lung transplant recipients with OB, neutrophilia is present and highly correlated with the presence of IL-8.

IL-12 is a proinflammatory cytokine that has recently been shown to have beneficial effects in the setting of acquired host immunity. To determine the role of IL-12 in innate immunity against Gram-negative bacterial organisms, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally (i.t.), resulting in the time-dependent expression of IL-12 mRNA (p35 and p40) and protein within the lung. Passive immunization of animals with anti-IL-12 serum i.p. at the time of K. pneumoniae inoculation resulted in a 12-fold increase in K. pneumoniae CFU in lung homogenates at 48 h, as compared with animals receiving control serum. In addition, treatment of Klebsiella-infected mice with anti-IL-12 Abs significantly decreased both short and long term survival. To assess the effect of compartmentalized IL-12 overexpression on outcome in Klebsiella pneumonia, animals were treated i.t. with 5 x 10(8) PFU of a nonreplicating adenoviral vector containing a human cytomegalovirus promoter and cDNAs coding for the p35 and p40 subunits of IL-12 inserted into the E1 and E3 domains (Ad5mIL-12), respectively. In vivo transfection with Ad5mIL-12 resulted in 45% long term survival in Klebsiella pneumonia, whereas no animals with Klebsiella pneumonia receiving control adenovirus survived. Moreover, treatment with anti-IFN-gamma Abs or soluble TNF receptor:Ig construct partially and completely attenuated survival benefits observed in animals receiving Ad5mIL-12, respectively. In conclusion, endogenous IL-12 is a critical component of antibacterial host defense, and the compartmentalized overexpression of IL-12 using recombinant adenoviral gene therapy represents a safe and effective approach to deliver IL-12 to the lung in the setting of murine Klebsiella pneumonia.

By 24 h after mechanical trauma to the cerebral cortex, astroglial reaction begins and injury sites are infiltrated by activated mononuclear phagocytes derived from blood-borne monocytes and endogenous microglia. There is little information about cellular interactions between astrocytes and leukocytes during this process. We previously showed that murine astrocytes produce chemokines including monocyte chemoattractant protein-1 (MCP-1) during experimental autoimmune encephalomyelitis. In this study, we asked whether astrocytes produce MCP-1 in the absence of immune mediated inflammation. To address this question, we analyzed the time course and cellular source of MCP-1 in mouse brain after penetrating mechanical injury, with particular focus on early time points before histologic detection of infiltrating mononuclear phagocytes. We observed sharply increased steady state levels of MCP-1 mRNA within 3 h after nitrocellulose membrane stab or implant injury to the adult mouse brain, and MCP-1 protein elevations were documented at 12 h postinjury. In situ hybridization combined with immunohistochemistry for the glial fibrillary acidic protein astrocyte marker showed that astrocytes were the cellular source of MCP-1 mRNA at these early time points after mechanical brain injury. Stab injury to the neonatal brain evoked neither MCP-1 expression nor astrogliosis. These results demonstrate that chemokine gene expression comprises one component of the astrocyte activation program. The data are consistent with a role for MCP-1 in the central nervous system inflammatory response to trauma.

The increased reactivity of mast cells during allergic airway inflammation has been linked to several aspects of pulmonary disease. A primary inducer of mast cell differentiation, proliferation, and activation has been identified as c-kit ligand or stem cell factor (SCF). In the present study, we used an established murine model of allergic eosinophilic airway inflammation to examine the role of SCF during an Ag-specific airway response. Initial data demonstrates increased SCF protein production at 8 h postchallenge in both lungs and serum of allergen-challenged, but not vehicle-challenged, mice. The immunolocalization of SCF in Ag-challenged lungs suggested that macrophage populations were the primary source of SCF, while epithelial cell regions also stained positive. Intense immunohistochemical staining of macrophages in bronchoalveolar lavage samples recovered from Ag-sensitized mice indicate that these cells may be a significant source of SCF in the lungs. Alveolar macrophages from the airways of normal mice stimulated with either TNF (0.1-10 ng/ml) or IL-4 (10 ng/ml) produced significant levels of SCF. Furthermore, neutralization studies demonstrated that the inhibition of airway SCF during allergen challenge significantly decreased eosinophil, but not neutrophil, infiltration throughout the response. Furthermore, when mice were treated with anti-SCF Ab, histamine levels were significantly reduced at 8 h postchallenge, the time of significant SCF production. Together, these data indicate that the production of SCF during Ag-induced lung inflammation by alveolar macrophages can play a significant role in the subsequent recruitment of eosinophils, possibly via mast cell activation and degranulation.

To determine whether human corneal epithelial cells and keratocytes produce chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES) protein and monocyte chemotactic protein-1 (MCP-1) after exposure to proinflammatory cytokines interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha).Pure cultures of human corneal epithelial cells and keratocytes were exposed to increasing concentrations of either human recombinant IL-1 alpha or TNF-alpha. At selected time intervals after exposure, culture supernatants were removed and assayed for RANTES and MCP-1 by the enzyme-linked immunosorbent assay. Total RNA was extracted from the cell cultures, and steady state mRNA levels were measured by reverse transcriptase-polymerase chain reaction.Exposure to keratocytes to either IL-1 alpha or TNF-alpha resulted in100-fold increases in RANTES protein secretion and150-fold increases in MCP-1 protein secretion, as well as in rapid sustained increases in intracellular levels of their corresponding mRNAs. Exposure of corneal epithelial cells to IL-1 alpha and TNF-alpha did not stimulate MCP-1 secretion nor intracellular levels of MCP-1 mRNA. Epithelial cells also failed to secrete RANTES protein even through the two inducing cytokines did stimulate increased expression of RANTES mRNA.These results suggest that RANTES and MCP-1 gene expression in human keratocytes differs markedly from their expression in human corneal epithelial cells and that the stroma of the cornea may be more important than the epithelium in recruitment of mononuclear leukocytes responsible for cell-mediated immunity.

Cryptococcus neoformans is acquired via the respiratory tract and is the leading cause of fatal mycosis in AIDS. Development of a T cell-mediated pulmonary inflammatory response is critical for clearance of this pathogen; however, the chemotactic factors that mediate inflammatory cell recruitment into the lungs have not been identified. In the present study, the bronchoalveolar lavage (BAL) fluid levels of the C-C chemokine monocyte chemotactic protein-1 (MCP-1) and the recruitment of inflammatory cells both increased following pulmonary infection with C. neoformans. The kinetics of MCP-1 production in the lungs correlated most closely with the recruitment of CD4+ T cells and monocytes/macrophages. Administration of neutralizing anti-MCP-1 Abs in vivo reduced the BAL fluid levels of MCP-1, decreased the recruitment of both macrophages (95%) and CD4+ T cells (76 +/- 9%), and inhibited cryptococcal clearance. Although no in vitro neutrophil or B cell chemotactic activity has been reported for MCP-1, recruitment of these leukocytes was also decreased in anti-MCP-1-treated mice (most likely an indirect effect of reducing the number of CD4+ T cells and macrophages). Neutralization of MCP-1 also resulted in decreased BAL fluid levels of TNF-alpha and IL-6. This is the first demonstration of a role for MCP-1 in clearance of an infection, and provides direct evidence that MCP-1 plays a critical role in the T cell-dependent immune response to C. neoformans.

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated, inflammatory demyelinating disease of the central nervous system (CNS) that serves as a model for the human demyelinating disease, multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific T lymphocytes and Ag-nonspecific mononuclear cells into the CNS. In the present report we investigated the role of two C-C chemokines (macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemotactic protein-1) and a C-x-C chemokine (MIP-2) in the pathogenesis of EAE. Production in the CNS of MIP-1 alpha, but not that of MIP-2, a rodent homologue of IL-8, or monocyte chemotactic protein-1, correlated with development of severe clinical disease. Administration of anti-MIP-1 alpha, but not that of anti-monocyte chemotactic protein-1, prevented the development of both acute and relapsing paralytic disease as well as infiltration of mononuclear cells into the CNS initiated by the transfer of neuroantigen peptide-activated T cells. Ab therapy could also be used to ameliorate the severity of ongoing clinical disease. Anti-MIP-1 alpha did not affect the activation of encepahlitogenic T cells as measured by cytokine secretion, surface marker expression, and ability to adoptively transfer EAE. These results demonstrate that MIP-1 alpha plays an important role in directing the chemoattraction of mononuclear inflammatory cells in the T cell-mediated autoimmune disease, EAE.

In bacterial meningitis, the recruitment of leukocytes across the blood-brain barrier into the central nervous system may be crucial for both elimination of pathogens and tissue injury. In addition to bacterial cell wall products, host factors including chemokines may lead to accumulation of phagocytes within the central nervous system. As shown by Northern analysis, brains of mice infected intracerebrally with Listeria monocytogenes (LM) express mRNA for three chemokines, the macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and MIP-2. The cellular sources of these chemokines comprise both the blood-derived polymorphonuclear leukocytes (PMNs) and monocytes infiltrating the meninges, the ventricular system and the periventricular area. In the course of meningitis a time-dependent increase of MIP-1 alpha and MIP-2 was found in the cerebrospinal fluid (CSF) by ELISA. CSF taken 24 h after infection (CSF-LM24) induced migration of human leukocytes when treated in chemotactic chambers in vitro. Neutralizing Abs to chemokines identified MIP-1 alpha and MIP-2 to be responsible for CSF-LM24 mediated chemotaxis of monocytes and PMNs, respectively. CSF obtained from mock-infected animals contained no MIP-1 alpha or MIP-2 and did not lead to migration of leukocytes. When testing CSF-LM24 on mouse spleen cells, the chemotactic activity detected for mononuclear cells was only partly inhibited by Abs to MIP-1 alpha and -1 beta. Thus, in addition to MIP-1 and -2 other not yet defined chemotactic factors are of importance for recruitment of leukocytes in bacterial meningitis.

Systemic exposure to LPS initiates a complex sequence of events resulting in organ-specific leukocyte recruitment and end-organ injury. We hypothesized that macrophage inflammatory protein-1 alpha (MIP-1 alpha), a C-C chemokine with leukocyte chemotactic and activating properties, may play an important role in lung inflammatory cell recruitment, subsequent lung injury, and mortality in endotoxemia. CD-1 mice were challenged with LPS (200 micrograms), resulting in a maximal 3.5-fold increase in neutrophils (polymorphonuclear leukocytes (PMNs)) at 6 h post-LPS, and a 2.6-fold increase in numbers of macrophages (M phi) within lung minces at 24 h. A time-dependent increase in MIP-1 alpha mRNA and protein was detected in lung after LPS treatment, with immunolocalization of MIP-1 alpha to blood and lung M phi, and the subendothelium. The pretreatment of mice with rabbit anti-MIP-1 alpha Ab resulted in a decrease in the influx of PMNs at 6 h, and influx of M phi at 24 h post-LPS challenge, an approximately 65% reduction in LPS-induced lung permeability to Evans blue, and a modest decrease in mortality at 24, but not 48 h post-LPS. Furthermore, passive immunization of mice with anti-MIP-1 alpha serum resulted in a 35% reduction in ICAM-1 mRNA levels within lung homogenates post-LPS. Finally, the pretreatment of animals with sTNFR:Fc (soluble TNF receptor:Ig construct) resulted in a 60% reduction in LPS-induced MIP-1 alpha mRNA expression within lung homogenates at 4 h post-LPS. Our studies indicate that MIP-1 alpha plays an integral role as a mediator of both PMN and M phi recruitment in murine endotoxemia.

Effective host defense against bacterial infection is dependent upon the vigorous recruitment and activation of neutrophils and macrophages. We hypothesized that IL-10 is produced in the setting of bacterial pneumonia, and this cytokine may attenuate host defense by inhibiting the expression of important activating and chemotactic cytokines. CD-1 mice were challenged with either 30 microliters of saline or saline containing 10(3) CFUs of Klebsiella pneumoniae intratracheally (i.t.) and lungs were harvested at 8, 24, and 48 h. The i.t. inoculation with K. pneumoniae resulted in a 13-, 14-, and 8-fold increase in lung homogenate TNF, macrophage inflammatory protein-2 (MIP-2), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) levels, respectively, as compared with control animals. In addition, we observed an increase in IL-10 mRNA and protein levels in lung homogenates, maximal at 48 h postinoculation. To establish the biologic relevance of IL-10 in Klebsiella pneumonia, we passively immunized CD-1 mice with 0.5 ml of rabbit anti-murine IL-10 serum or preimmune serum i.p. 2 h before i.t. administration of K. pneumoniae. Treatment of animals with anti-IL-10 serum resulted in increased levels of TNF, MIP-2, and MIP-1 alpha, respectively, within lung homogenates at 24 and 48 h, as compared with preimmune-treated animals. Furthermore, neutralization of IL-10 resulted in a significant decrease in K. pneumoniae CFU in both lung homogenates and plasma harvested at 48 h, as well as a significant increase in survival in these animals. Our studies indicate that 1) IL-10 is produced during Klebsiella pneumonia; and 2) inhibition of IL-10 bioactivity in vivo results in enhanced bacterial clearance, increased expression of proinflammatory cytokines, and prolonged survival.

Airway inflammation is characterized by leukocyte extravasation around the peribronchial mucosa and into the airway of the lung. In the present study we utilized a model of airway inflammation induced by intratracheal challenge with soluble parasite (Schistosoma mansoni) egg Ag (SEA) in presensitized mice. The subsequent inflammatory response and leukocyte recruitment consists of early neutrophil (8 to 24 h) and later eosinophil (48 to 72 h) infiltration into the interstitium and airway. Little neutrophil and no eosinophil recruitment was observed in presensitized control mice challenged with vehicle. Multiple studies have demonstrated a crucial role for TNF-alpha during inflammatory responses. In these experiments we investigated the role of TNF-alpha in Ag-specific eosinophilic airway inflammation. Measurement of TNF-alpha expression by reverse transcriptase-PCR and ELISA in whole lung homogenates of SEA-challenged mice demonstrated an early increase in TNF-alpha levels (1 to 8 h). To determine the specific role of TNF-alpha in leukocyte recruitment during airway inflammation, mice were treated with soluble TNF-alpha receptor linked to an Fc Ab molecule (sTNFr-:Fc). This treatment has previously been used to effectively neutralize TNF in vivo. Intratracheal SEA-challenged mice treated with sTNFr-FC demonstrated significantly decreased leukocyte recruitment into the lung and airway. The inflammatory response in the lungs in sTNFr-Fc-treated mice was significantly decreased throughout the study period, as compared with control mice. An approximate decrease in early neutrophil infiltration into the airway was observed when sTNFr-Fc was administered 2 h before the Ag challenge. Eosinophil infiltration was also diminished when sTNFr-Fc was administered before Ag challenge. Interestingly, when sTNFr-Fc was administered therapeutically 24 h after Ag challenge, the eosinophil response was nearly abrogated at 48 h after challenge. These studies indicate that TNF-alpha acts as an initial inflammatory cytokine that subsequently regulates both early neutrophil infiltration and eosinophil recruitment into the lung and airspace.

Systemic exposure to LPS initiates a complex sequence of events culminating in organ-specific leukocyte recruitment and end organ injury. We hypothesized that RANTES, a C-C chemokine with potent M phi (mononuclear phagocyte) chemotactic activity, is expressed in vivo in response to endotoxemia, and that this protein may play an important role in the recruitment of M phi to the lung. CD-1 mice were challenged with LPS (200 micrograms), resulting in a maximal fourfold increase in polymorphonuclear leukocyte (neutrophils) at 6 h post LPS, and a 2.4-fold increase in numbers of M phi within lung minces at 24 h. A time-dependent increase in RANTES mRNA was detected in lung after LPS treatment, whereas minimal quantities of RANTES mRNA were detected in blood buffy coats and liver. Furthermore, treatment with LPS resulted in time-dependent increase in RANTES protein within lung homogenates, with immunolocalization to alveolar epithelial cells. The pretreatment of mice with goat anti-RANTES Ab significantly inhibited the influx of lung M phi, but not polymorphonuclear leukocyte and lymphocytes, at 24 h post-LPS challenge. Lastly, the pretreatment of animals with soluble TNF receptor: Ig construct 2 h before LPS resulted in a 60% reduction in steady state levels of RANTES mRNA within lung homogenates at 4 h post-LPS. Our observations suggest that RANTES represents an important mediator of lung M phi recruitment in the setting of endotoxemia, and that the expression of RANTES in vivo is dependent upon the endogenous production of TNF.

The present study explored the role of murine monocyte chemotactic protein (MCP) in the T cell-mediated hypersensitive granulomatous response to Schistosoma mansoni eggs. The study examined the time course of local production, contribution to cellular infiltration, and the role of T cells in endogenous regulation. Synchronized pulmonary granulomas were induced under conditions of primary and secondary states of immunity. Primer-directed polymerase chain reaction analysis showed increased MCP mRNA expression in granulomatous lungs, mainly in the secondary response. Levels of MCP were measured by enzyme-linked immunosorbent assay in cultures of intact granulomas. Spontaneous MCP production was modest in primary granuloma cultures, reaching a maximum of 5.7 +/- 0.9 ng/ml by 16 days. In contrast, the secondary response showed augmented and accelerated production, achieving 13 +/- 2.0 ng/ml by 2 days. Immunohistochemical staining revealed the strongest MCP expression within microvascular adventitial cells or pericytes as well as in scattered mononuclear cells associated with granulomas. Staining was not detected in normal lungs. Passive immunization with anti-MCP-1 antibodies caused a 40% reduction in the secondary granuloma area but did not significantly affect the primary response. With adoptive cell transfer and T cell subset depletion, it was shown that Thy-1+ and CD5+ cells augmented, whereas CD8+ cells appeared to impair, MCP production. This provides direct evidence that MCP is involved in secondary Th2-mediated response to schistosome eggs and is subject to regulation by T cells.

Recent studies have demonstrated a crucial role for TNF during inflammatory granuloma formation. In addition, TNF has been shown to up-regulate adhesion molecules that participate in cellular recruitment and lymphocyte activation. In the present study, we have examined the mechanism of TNF activation during Schistosoma mansoni egg granuloma formation and its relationship to the expression of ICAM-1. Our initial studies showed that high affinity human soluble TNFR coupled to the Fc portion of an Ig (sTNFR:Fc construct) could effectively diminish granuloma formation and lymphocyte activation in vivo. We have also assessed the increased expression of ICAM-1, its contribution to granuloma development, and its relationship with TNF during lesion formation. Increased steady state ICAM-1 mRNA expression was observed in primary egg granulomas when compared with normal lung and foreign body (Sephadex bead) granulomas, which suggests a role for ICAM-1 in Ag-induced lesion formation. Subsequent studies have demonstrated that sTNFR:Fc treatment down-regulated granuloma formation and ICAM-1 expression, thus suggesting one mechanism of TNF involvement in granuloma formation was through the induction of ICAM-1. Anti-ICAM-1 decreased the soluble egg Ag-specific T cell proliferation in vitro. In addition, passive immunization of mice with anti-ICAM-1 mAb during primary granuloma formation resulted in an attenuation of lesion development as compared with lesion development in a control Ab-treated group. The proliferative response to soluble egg Ag was also significantly reduced in ex vivo experiments that used spleen cells from the anti-ICAM-1 treated mice. These data demonstrate that both TNF and ICAM-1 participate in lymphocyte activation and granuloma formation and suggest that one mechanism of TNF in granuloma development is through TNF-induced ICAM-1 expression.

The formation of hepatic granulomas around persistently deposited Schistosoma mansoni eggs leads to parenchymal damage, ongoing fibrosis, and ultimate loss of liver function. In this study, the production of macrophage inflammatory protein-1 alpha (MIP-1) and monocyte chemoattractant protein-1 (MCP-1) by granuloma fibroblasts was examined to establish the potential contribution of intragranuloma fibroblasts to the maintenance of the chronic inflammation. Isolated fibroblasts from dispersed acute infection hepatic granulomas were grown in tissue culture for 3 to 4 weeks and used on the third or fourth passage. We initially surveyed fibroblasts for production of MIP-1 and MCP-1 by reverse transcription-polymerase chain reaction (RT-PCR) after stimulation with interleukin (IL)-1, tumor necrosis factor, interferon (IFN)-gamma, IL-4, or IL-10: cytokines found within the granuloma. These studies demonstrated constitutive expression of MCP-1 and differential up-regulation of MIP-1 on cytokine stimulation. Protein expression was then verified by immunohistochemical localization of MIP-1 and MCP-1 in paraformaldehyde-fixed fibroblasts and by direct quantitation of MIP-1 and MCP-1 in culture supernatants by specific ELISAs. These studies demonstrated constitutive expression of MCP-1 in unstimulated and cytokine-stimulated granuloma fibroblasts. In contrast, IL-1 (0.1 to 2.5 ng/ml), IFN-gamma (10 micrograms/ml), and IL-10 (2.5 to 10 ng/ml) were able to induce the significant production of MIP-1 by the granuloma fibroblasts. Interestingly, normal noninflammatory fibroblasts from uninfected mice showed no significant production of MIP-1 or MCP-1 in response to these cytokines. These results suggest that granuloma fibroblasts may be phenotypically altered compared with normal fibroblasts and have a significant role in leukocyte recruitment, granuloma growth, and maintenance of the egg-induced lesion.

Mononuclear phagocyte (M phi) recruitment and activation is a hallmark of a number of chronic inflammatory diseases of the lung, including sarcoidosis and idiopathic pulmonary fibrosis (IPF). We hypothesized that macrophage inflammatory protein-1 (MIP-1 alpha), a peptide with leukocyte activating and chemotactic properties, may play an important role in mediating many of the cellular changes that occur in sarcoidosis and IPF. In initial experiments, we demonstrated that human rMIP-1 alpha exerted chemotactic activities toward both polymorphonuclear leukocytes and monocytes, and these activities were inhibited by treatment with rabbit anti-human MIP-1 alpha antiserum. In support of the potential role of MIP-1 alpha in interstitial lung disease, we detected MIP-1 alpha in the bronchoalveolar lavage fluid of 22/23 patients with sarcoidosis (mean 443 +/- 76 pg/ml) and 9/9 patients with IPF (mean 427 +/- 81 pg/ml), whereas detectable MIP-1 alpha was found in only 1/7 healthy subjects (mean 64 +/- 64 pg/ml). In addition, we found a 2.5- and 1.8-fold increase in monocyte chemotactic activity in BALF obtained from patients with sarcoidosis and IPF respectively, as compared to healthy subjects, and this monocyte chemotactic activity, but not neutrophil chemotactic activity, was reduced by approximately 22% when bronchoalveolar lavage fluid from sarcoidosis and IPF patients were preincubated with rabbit antihuman MIP-1 alpha antibodies. To determine the cellular source(s) of MIP-1 alpha within the lung, we performed immunohistochemical analysis of bronchoalveolar lavage cell pellets, transbronchial biopsies, and open lung biopsies obtained from patients with IPF and sarcoidosis. Substantial expression of cell-associated MIP-1 alpha was detected in M phi, including both alveolar AM phi and interstitial M phi. In addition, interstitial fibroblasts within biopsies obtained from sarcoid and IPF patients also expressed immunoreactive MIP-1 alpha. Minimal to no detectable MIP-1 alpha was expressed in alveolar M phi from healthy subjects or interstitial cells in lung biopsy specimens obtained from patients undergoing thoracotomy for malignancy. Furthermore, pulmonary fibroblasts isolated from patients with IPF produced greater amounts of MIP-1 alpha after challenge with IL-1 beta than did similarly treated pulmonary fibroblasts recovered from patients without fibrotic lung disease. Our findings suggest that MIP-1 alpha is expressed in increased amounts within the airspace and interstitium of patients with sarcoidosis and IPF, and that this cytokine may be an important mediator of both M phi activation and recruitment that characterize these disease states.

Cytokines and cellular adhesion molecules (CAMs) may play a role in the inflammatory and fibrotic processes underlying systemic sclerosis (SSc). We compared the immunohistological distribution of cytokines and CAMs in skin biopsies from 12 SSc patients and 14 normal (NL) individuals. Among CAMs, vascular cell adhesion molecule-1 (VCAM-1), which mediates leukocyte-endothelial adhesion, showed increased expression on SSc versus NL endothelium and stratum granulosum. P-selectin was up-regulated in SSc versus NL stratum granulosum. The CD44 lymphocyte homing receptor showed the most striking differences between SSc and NL: its expression was increased in SSc stratum granulosum, stratum spinosum, on lymphocytes, and macrophages. Regarding cytokines, interleukin-6 (IL-6) expression was increased on SSc versus NL endothelium and fibroblasts. Tumor necrosis factor-alpha (TNF-alpha) reactivity was more prevalent in SSc than NL stratum granulosum, whereas IL-8 expression was higher on SSc compared to NL endothelium. Some CAMs, such as VCAM-1 and P-selectin, and cytokines, namely TNF-alpha and IL-8, were more commonly found in skin biopsies taken from early (or = 1 year's duration) SSc, while others, such as IL-6, showed up-regulation in the late stage of the disease. The results suggest that certain CAMs and cytokines may play a differential role in both the early, inflammatory, and the late, fibrotic stage of SSc.

Patients suffering from serious bacterial infection present to the hospital after early inflammatory events, such as release of tumor necrosis factor (TNF), have been initiated. The role of other cytokines, such as interleukin-8 (IL-8), a neutrophil chemoattractant and activator, in the pathophysiology of human sepsis is not well characterized, and there are only limited data on IL-6. We studied serial concentrations of TNF, IL-6 (involved in the acute-phase response), and IL-8 in plasma and leukocyte levels of mRNA for these cytokines in patients with localized and septicemic Pseudomonas pseudomallei infection on admission to the hospital and during a prolonged recovery phase (up to 30 days). Of 18 patients, 8 had detectable plasma IL-8 and all had raised plasma IL-6 concentrations. In patients who died median initial concentration of IL-8 (167 pg/ml; range, 97 to 362 pg/ml) and IL-6 (4,800 pg/ml; range, 60 to 9,245 pg/ml) in plasma were higher than those in survivors (P less than 0.008 and P = 0.007, respectively). Septic patients who survived and patients with localized disease had similar cytokine levels. Plasma IL-8 and IL-6 concentrations were elevated throughout the inpatient period of recovery. Circulating leukocytes contained mRNA for IL-8 but not for IL-6 and TNF, and they may secrete IL-8. An elevated plasma IL-6 concentration (greater than 1,000 pg/ml) had 75% mortality) was the best predictor of mortality in P. pseudomallei sepsis. Fifty percent of patients with detectable plasma IL-8 concentrations died. In contrast, plasma TNF bioactivity did not relate to outcome; 75% of patients who did never had detectable plasma TNF activity.

PGE2, an immune mediator, is an inhibitor of LPS-stimulated TNF production and gene transcription. In the present study we determined whether pretreatment with PGE2 could desensitize the suppressive function of PGE2 for the production of macrophage (MO)-derived TNF. CFA-elicited MO were incubated with PGE2 or medium only, washed, and then challenged with graded doses of LPS (0.001 to 1000 ng/ml) in the presence or absence of new PGE2. The concomitant addition of PGE2 with LPS shifted the LPS concentration-effect curve 16-fold to the right with a 52% decrease in the maximum LPS response, while MO pretreated with PGE2, washed, and incubated with LPS plus new PGE2 were desensitized to TNF regulation. These latter conditions resulted in a complete loss of the ability of PGE2 to inhibit MO TNF production as demonstrated by no significant change in the EC50 of LPS. In addition, the PGE2 concentration effect curve was shifted to the right after pretreatment of MO, suggesting a desensitized PGE2 receptor system. At the transcriptional level, pretreatment of MO with PGE2 attenuated the ability of new PGE2 to inhibit LPS-dependent TNF mRNA expression. Further studies demonstrated that, although the concomitant addition of the cyclooxygenase inhibitor indomethacin plus LPS could increase TNF production, MO pretreated with indomethacin, washed, and then challenged with LPS demonstrated an inhibition of TNF expression. MO pretreated with indomethacin also demonstrated an increased sensitivity for exogenous PGE2-induced suppression of TNF mRNA and bioactivity. These investigations further support the role of PGE2 as an immunomodulating compound that may effectively regulate the local concentration of specific monokines needed to maintain an inflammatory lesion.