Novel protective effects of stem cell factor in a murine model of acute septic peritonitis. Dependence on MCP-1.

Mast cells participate in the host response during sepsis and have been shown to have a protective effect in a murine model of acute septic peritonitis and multi-organ failure initiated by cecal ligation and puncture (CLP). Stem cell factor (SCF) is a hematopoietic cytokine important in mast cell proliferation and activation. In the present study, we examined the protective effects of a single intraperitoneal injection of SCF given 2 hours before CLP surgery in mice. Four days after the CLP surgery, SCF pretreatment significantly improved mouse survival from 29 to 56% and mast cells were absolutely required for this effect. Immunoneutralization studies revealed that the SCF-stimulated release of monocyte chemoattractant protein-1 (MCP-1) into the septic peritoneal cavity contributed to the protective effect of SCF in this model. One potential cellular source of MCP-1 was the SCF-activated mast cell. In addition, SCF pretreatment significantly augmented circulating levels of SCF and the immunomodulatory cytokine interleukin-10 in septic mice, in part because the SCF pretreatment seemed to promote the release of both mediators from the liver. Additional hepatic effects of SCF treatment included an accelerated expression of hepatic levels of signal transducer and activator of transcription-3 (STAT-3) in CLP mice pretreated with SCF. Taken together, the findings from the present study demonstrate that the intraperitoneal delivery of SCF has a major protective effect in a murine model of CLP.

Chemokine C10 promotes disease resolution and survival in an experimental model of bacterial sepsis.

Previous studies have suggested that the C-C chemokine C10 is involved in the chronic stages of host defense reactions. The present study addressed the role of C10 in a murine model of septic peritonitis, induced by cecal ligation and puncture (CLP). Unlike other C-C chemokines, C10 levels in the peritoneal wash were increased approximately 30-fold above baseline levels at 48 h after CLP surgery. Immunoneutralization of peritoneal C10 levels with polyclonal anti-C10 antiserum during CLP-induced peritonitis negatively impacted mouse survival over 4 days. In contrast, when 500 ng of recombinant murine C10 was administered immediately after CLP surgery, the 4-day survival rate increased from 20% to over 60%. The C10 therapy appeared to facilitate a rapid and significant enhancement of the levels of tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1) and a later increase in interleukin-13 (IL-13) levels in the peritoneal cavity. In vitro studies showed that the combination of IL-1beta and C10 markedly augmented TNF-alpha synthesis by peritoneal macrophages and that C10 synthesis was induced in these cells following their exposure to IL-13. At 24 h after CLP surgery, only 25% of C10-treated mice were bacteremic versus 85% of the control group that exhibited dissemination of bacteria into the circulation. The lack of bacteremia in C10-treated mice appeared to be related, in part, to in vitro evidence that C10 significantly enhanced the bacterial phagocytic activity of peritoneal macrophages. In addition, in vivo evidence suggested that C10 therapy significantly reduced the amount of material that leaked from the damaged gut. Taken together, the results of this study demonstrate that the C10 chemokine rapidly promotes disease resolution in the CLP model through its direct effects on the cellular events critically involved in host defense during septic peritonitis.

Therapeutic use of chemokines.

Chemokines are involved in a number of pathological processes, and therefore represent important targets. However, it has also become apparent that chemokines have exciting therapeutic applications in inflammatory, infectious and cancer-related diseases. The following review will highlight the application of novel therapies including viral-encoded, recombinant, and genetically engineered chemokines to a number of diseases or disorders. Advances in the application of novel chemokine delivery procedures both at the research bench and the clinical bedside will also be discussed. Overall, the utilization of chemokines to prevent and treat disease has tremendous potential.

Exaggerated hepatic injury due to acetaminophen challenge in mice lacking C-C chemokine receptor 2.

Monocyte chemoattractant protein-1 is one of the major C-C chemokines that has been implicated in liver injury. The C-C chemokine receptor, CCR2, has been identified as the primary receptor that mediates monocyte chemoattractant protein-1 (MCP-1) responses in the mouse. Accordingly, the present study addressed the role of CCR2 in mice acutely challenged with acetaminophen (APAP). Mice genetically deficient in CCR2 (CCR2(-/-)) and their wild-type counterparts (CCR2(+/+)) were fasted for 10 hours before receiving an intraperitoneal injection of APAP (300 mg/kg). Liver and serum samples were removed from both groups of mice before and at 24 and 48 hours post APAP. Significantly elevated levels of MCP-1 were detected in liver samples from CCR2(+/+) and CCR2(-/-) mice at 24 hours post-APAP. Although CCR2(+/+) mice exhibited no liver injury at any time after receiving APAP, CCR2(-/-) mice exhibited marked evidence of necrotic and TUNEL-positive cells in the liver, particularly at 24 hours post-APAP. Enzyme-linked immunosorbent assay analysis of liver homogenates from both groups of mice at the 24 hours time point revealed that liver tissue from CCR2(-/-) mice contained significantly greater amounts of immunoreactive IFN-gamma and TNF-alpha. The in vivo immunoneutralization of IFN-gamma or TNF-alpha significantly attenuated APAP-induced liver injury in CCR2(-/-) mice and increased hepatic IL-13 levels. Taken together, these findings demonstrate that CCR2 expression in the liver provides a hepatoprotective effect through its regulation of cytokine generation during APAP challenge.

Chronic airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis during allergic airway disease induced by Aspergillus fumigatus.

Clinical allergic airway disease is associated with persistent airway hyperreactivity and remodeling, but little is known about the mechanisms leading to these alterations. This paucity of information is related in part to the absence of chronic models of allergic airway disease. Herein we describe a model of persistent airway hyperreactivity, goblet cell hyperplasia, and subepithelial fibrosis that is initiated by the intratracheal introduction of Aspergillus fumigatus spores or conidia into the airways of mice previously sensitized to A. fumigatus. Similar persistent airway alterations were not observed in nonsensitized mice challenged with A. fumigatus conidia alone. A. fumigatus-sensitized mice exhibited significantly enhanced airway hyperresponsiveness to a methacholine challenge that was still present at 30 days after the conidia challenge. Eosinophils and lymphocytes were present in bronchoalveolar lavage (BAL) samples from A. fumigatus-sensitized mice at all times after conidia challenge. Compared with levels measured in A. fumigatus-sensitized mice immediately before conidia, significantly elevated interferon-gamma (IFN-gamma) and transforming growth factor (TGF-beta) levels were present in whole lung homogenates up to 7 days after the conidia challenge. At day 30 after conidia challenge, significantly elevated levels of interleukin-4 (IL-4) and IL-13 were present in the A. fumigatus-sensitized mice. Histological analysis revealed profound goblet cell hyperplasia and airway fibrosis at days 30 after conidia, and the latter finding was confirmed by hydroxyproline measurements. Thus the introduction of A. fumigatus conidia into A. fumigatus-sensitized mice results in persistent airway hyperresponsiveness, fibrosis, and goblet cell hyperplasia.

Macrophage inflammatory protein-2 gene therapy attenuates adenovirus- and acetaminophen-mediated hepatic injury.

Profound hepatocellular injury is often a consequence of adenovirus-mediated gene therapy or acetaminophen ingestion. The aim of the present study was to examine the role of a CXC chemokine, macrophage inflammatory protein-2 (MIP-2), in the hepatotoxic response by mice infected with adenovirus and challenged with acetaminophen. CD1 mice that received a replication-defective human type 5 adenovirus vector (Ad70-3) intravenously exhibited hepatic injury that peaked at 24 h after infection. In contrast, mice that received a similar adenovirus vector containing a rodent MIP-2 cDNA insert had no hepatic injury at any time after infection. The combination of Ad70-3 infection and an intraperitoneal challenge with 400 mg/kg of acetaminophen was fatal in 50% of the mice, but only 10% of the AdMIP-2 group receiving acetaminophen were similarly affected. Furthermore, AdMIP-2 mice had significantly lower hepatic injury and serum aminotransaminases compared with the Ad70-3 group. However, AdMIP-2 infection in mice lacking the CXC chemokine receptor that binds MIP-2, CXCR2, did not attenuate any of the markers of liver injury after adenovirus and acetaminophen challenge. AdMIP-2 treatment of CD1 mice was also associated with significantly decreased leukocyte infiltration into the liver and an earlier increase in hepatic 3H-thymidine incorporation compared with the control group. Taken together, these data demonstrate that MIP-2 has a protective role in both adenovirus- and acetaminophen-mediated hepatotoxicity, and suggest that MIP-2 may promote rapid hepatic regeneration following acute hepatic injury.

Novel CXCR2-dependent liver regenerative qualities of ELR-containing CXC chemokines.

Severe acute liver injury due to accidental or intentional acetaminophen overdose presents a major clinical dilemma often requiring liver transplantation. In the present study, liver regeneration after profound liver injury in mice challenged with acetaminophen was facilitated by the exogenous addition of ELR-containing CXC chemokines such as macrophage inflammatory protein-2 (MIP-2), epithelial neutrophil-activating protein-78 (ENA-78), or interleukin 8. Intravenous administration of ELR-CXC chemokines or N-acetyl-cysteine (NAC) immediately after acetaminophen challenge in mice significantly reduced histological and biochemical markers of hepatic injury. However, when the intervention was delayed until 10 h after acetaminophen challenge, only ELR-CXC chemokines significantly reduced liver injury and mouse mortality. The delayed addition of ELR-CXC chemokines to cultured hepatocytes maintained the proliferation of these cells in a CXCR2-dependent fashion after acetaminophen challenge whereas delayed NAC treatment did not. These observations demonstrate that ELR-CXC chemokines represent novel hepatic regenerative factors that exhibit prolonged therapeutic effects after acetaminophen-induced hepatotoxicity.

Multiple roles for IL-12 in a model of acute septic peritonitis.

The present study addressed the role of IL-12 in a murine model of septic peritonitis, induced by cecal ligation and puncture (CLP). Although CLP surgery induced IL-12 production at 6 and 24 h after surgery, IL-12 immunoneutralization was clearly deleterious in this model: 54% of CLP mice receiving preimmune serum survived, whereas mice administered IL-12 antisera prior to CLP experienced a 25% survival rate. IL-12 immunoneutralization not only led to increased mortality, but also appeared to promote a shift away from IL-12 and IFN-gamma, in favor of IL-10. This cytokine shift corresponded to changes in bacterial load, as CLP mice receiving IL-12 antiserum yielded more CFUs from the peritoneal cavity at 24 h after CLP. To address the role of bacterial infection in IL-12 antiserum-induced mortality following CLP, antibiotics were administered for 4 days after surgery. Despite regular antibiotic administration, IL-12 immunoneutralization still reduced survival in CLP mice. Furthermore, histology of the ceca revealed that mice administered IL-12 antisera failed to show typical organization of the damaged cecum wall. Accordingly, Gram staining revealed bacteria within peritoneal fluids from these mice, while peritoneal fluids from CLP mice that received preimmune serum and antibiotics were free of bacteria. Altogether, these data suggested multiple important roles for IL-12 in the evolution of murine septic peritonitis.

IL-10 is a major mediator of sepsis-induced impairment in lung antibacterial host defense.

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.

Novel roles for chemokines and fibroblasts in interstitial fibrosis.

BACKGROUND: Regardless of its involvement in either wound healing or excessive fibrosis, the interstitial fibroblast can now be considered an important early participant in inflammatory responses. Although it is recognized that certain immune cells and proinflammatory mediators are intricately linked to fibrotic disease, little is presently known about the manner in which these mediators and cells are orchestrated to a fibrotic finale. Experimental studies have shown that interstitial fibroblasts are capable of participating in an inflammatory response by promoting direct fibroblast-to-immune cell communication and/or modulating the release of soluble mediators that are mutually recognized by both types of cells. METHODS: Primary cultures of murine fibroblasts were recovered from either normal tissue or tissue undergoing a cell-mediated inflammatory response. These stromal cells were assessed for the expression of various cytokines and chemokines indicative of a type 1 or type 2 response. In addition, the fibroblasts were co-cultured with mononuclear cells to assess the cell-to-cell communication. RESULTS: Fibroblasts recovered from different cell-mediated inflammatory responses demonstrated a dramatic alteration in their cytokine profile. Fibroblasts recovered from the type 2 immune response produced high levels of monocyte chemotactic protein-1 (MCP-1), as compared to the normal fibroblasts and fibroblasts recovered from the type 1 lesion. Mononuclear cells co-cultured with fibroblasts induced a contact-dependent expression of elevated levels of chemokines, especially the macrophage-derived MIP-1 alpha. Thus, both fibroblasts themselves and fibroblasts co-cultured with immune-inflammatory cells have the ability to participate in the maintenance of an inflammatory response via the expression of chemokines. CONCLUSIONS: Our laboratory and others have addressed the role of chemotactic cytokines or chemokines in the fibrotic process, and have demonstrated that fibroblasts are capable of modulating the activation of various immune cells that have been implicated in fibrotic disease. In addition, the interstitial fibroblast is capable of regulating its own behavior within the interstitial environment via the expression of chemokines and chemokine receptors. Thus, novel strategies aimed at preventing fibrotic disease will likely need to address the early engagement of inflammatory cells by fibroblasts, and possibly modulate the ability of fibroblasts to generate and/or recognize profibrotic signals supplied by chemokines.

Macrophage/fibroblast coculture induces macrophage inflammatory protein-1alpha production mediated by intercellular adhesion molecule-1 and oxygen radicals.

This study examined the cell-to-cell interaction between fibroblasts and macrophages as a possible contributor to the chronic inflammatory state. In a coculture system, consisting of macrophages layered over confluent fibroblasts, there was a significant increase in macrophage inflammatory protein 1alpha (MIP-1alpha) compared with control cultures. ICAM-1 adhesion was identified as an important stimulus of MIP-1alpha production by using ICAM-1-specific monoclonal antibodies. Furthermore, fibroblasts from ICAM-1 knockout mice induced significantly less MIP-1alpha production from peritoneal macrophages when compared to control fibroblasts. In addition, it appeared that oxygen radicals functioned as activating molecules during cellular interaction and production of MIP-1alpha, as the addition of the antioxidant N-acetylcysteine (NAC) prevented MIP-1alpha secretion. Thus, the ICAM-1 and oxygen radical-mediated induction of MIP-1alpha associated with a macrophage/fibroblast coculture system provides one possible mechanism by which immune/inflammatory cell interactions may augment chemokine production and exacerbate chronic inflammatory diseases.

Therapeutic effects of nitric oxide inhibition during experimental fecal peritonitis: role of interleukin-10 and monocyte chemoattractant protein 1.

This study demonstrates that the therapeutic effect of a nitric oxide inhibitor in a murine model of fecal peritonitis is mediated in part by increased levels of interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1). Female CD1 mice were subjected to cecal ligation and puncture (CLP) with a 21-gauge needle and, immediately following surgery, were injected intraperitoneally with saline, N(G)-nitro-L-arginine methyl ester (L-NAME; 8 mg/kg), or N(G)-nitro-D-arginine methyl ester (D-NAME; 8 mg/kg). At 96 h after surgery and drug treatment, 20% of mice that received D-NAME had survived whereas 60% of mice that received L-NAME were alive. To elucidate the effect of L-NAME treatment on chemokine and cytokine production during fecal peritonitis, the levels of macrophage inflammatory protein 2 (MIP-2), IL-10, and MCP-1 were measured in peritoneal washings from additional groups of mice 24 h after the CLP surgery. Peritoneal fluids from L-NAME-treated mice contained significantly higher levels of IL-10 and MCP-1 than did those from D-NAME-treated mice. To elucidate the effect of nitric oxide inhibition on potential cellular sources of IL-10 and MCP-1 in the CLP model, cultured alveolar and peritoneal macrophages were activated with bacterial lipopolysaccharide in the presence of L-NAME; these macrophages produced significantly more MCP-1 than did similarly activated macrophages in the presence of D-NAME. In the CLP surgery model, immunoneutralization of IL-10 alone or IL-10 and MCP-1 together with polyclonal antibodies prior to surgery significantly reduced the survival rates in L-NAME-treated groups compared with L-NAME-treated groups that received preimmune serum. Taken together, these data demonstrate that the inhibition of nitric oxide following experimental CLP fecal peritonitis is therapeutic, in part through the modulatory effect of this treatment on the synthesis of IL-10 and MCP-1.

Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide.

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.