Nickolas W. Lukacs

The role of chemokines in inflammatory joint disease

Rheumatoid arthritis (RA) is an autoimmune disease characterized by the elicitation and activation of a number of leukocyte populations within both the synovial space and joint tissue. The recruited leukocytes subsequently play an instrumental role in synovial cell proliferation, pannus formation, and bone erosion. Although it is known that leukocytes are important participants in the evolving joint pathology, the mechanism responsible for the successful elicitation of cells to the joint is not clear. A number of studies have identified an association of specific cytokines, including chemokines, with active arthritis, but longitudinal analyses of cytokine expression and the causal role of these mediators have not been defined. Animal models and cell culture systems have proved useful in identifying the expression of various cytokines during the maintenance of chronic joint inflammation. In addition, animal models have provided important information regarding the kinetic production and contribution of specific mediators to the development of experimental arthritis. These studies provide insight into the potential mechanisms for leukocyte involvement in inflammatory joint disease.

Expression and Contribution of Endogenous IL-13 in an Experimental Model of Sepsis

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

Activation and regulation of chemokines in allergic airway inflammation.

Allergic airway inflammation is characterized by peribronchial eosinophil accumulation within the submucosa surrounding the airway. The initial induction of immunoglobulin E (IgE)‐mediated mast cell degranulation, up‐regulation of adhesion molecules, and the production of inflammatory and chemotactic cytokines, leading to the infiltration of specific leukocyte subsets, is orchestrated in a sequential manner. The activation and degranulation of local mast cell populations is an immediate airway response mediated both by antigen‐specific, surface bound IgE and by cytokine‐induced activational pathways. Subsequently the infiltration and activation of effector leukocytes (neutrophils and eosinophils) mediated by the persistant activation of allergen‐specific T cells leads to pathological manifestations within the lung and airway. The development of appropriate animal models to dissect the critical mechanisms involved in antigen‐induced airway pathology is crucial for the development of efficacious therapies. We have utilized a model of allergic airway inflammation induced by intratracheal challenge with parasite (Schistosoma mansoni) egg antigen in presensitized mice. This model has proven useful in the assessment of eosinophil recruitment and has identified key cytokines involved in leukocyte elicitation. These cytokines include interleukin‐4 and tumor necrosis factor, which appear to act as early response mediators, as well as C‐C chemokines, macrophage inflammatory protein‐1a, and RANTES, which act directly on eosinophil recruitment. In addition, we have found that both C‐X‐C and C‐C chemokines are expressed in pulmonary‐derived mast cells, suggesting an important contribution to leukocyte responses in the allergic airway.

Pivotal Role of the CC Chemokine, Macrophage-Derived Chemokine, in the Innate Immune Response

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

Cell-to-cell and cell-to-matrix interactions mediate chemokine expression: an important component of the inflammatory lesion.

Although many studies have characterized soluble factors that stimulate or inhibit chemokine secretion, in this review we focus on the event of cellular adhesion as a novel mechanism for stimulating chemokine expression. Recent work has demonstrated chemokine expression following cell‐to‐cell and cell‐to‐matrix adhesion. The specificity of this finding was demonstrated utilizing various techniques that illustrate that adhesion, and not a soluble stimulus, is in some cases responsible for initiating or augmenting chemokine expression. For example, co‐cultures of peripheral blood monocytes and endothelial cells secreted elevated levels of IL‐8 and MCP‐1 compared with either cell type alone. When co‐cultured in transwells, this effect was significantly attenuated. In other experiments, neutralizing monoclonal antibodies to various adhesion molecules inhibited chemokine expression. The effects of adhesion were not limited to leukocytes. Both immune and non‐immune cell types were evaluated as potential sources of adhesion‐mediated chemokine expression. Not suprisingly, expression of some chemokines was associated with adhesion, whereas others were not, supporting the notion that adhesion differentially signals chemokine secretion during the inflammatory response. We hypothesize that as a recruited leukocyte encounters different adhesion substrates such as endothelial cells, basement membrane, extracellular matrix, and fibroblasts, the expression of chemokines from both the leukocyte and the substrate may be initiated, inhibited, or augmented. Careful characterization of the contribution of adhesion to regulation of chemokine expression will provide insight into the pathogenesis of many human diseases where chemokines have a central role. J. Leukoc. Biol.62: 612–619; 1997.

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.

Chemokine C10 Promotes Disease Resolution and Survival in an Experimental Model of Bacterial Sepsis

ABSTRACT 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-α) 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-1β and C10 markedly augmented TNF-α 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.

The role of interleukin-8 in the infectious process.

The recruitment of blood-born leukocytes to a specific site of infection is one of the most fundamental of all inflammatory processes. To successfully elicit the appropriate leukocyte populations, a number of dynamic alterations must first occur in a tightly coordinated manner. The first changes that are evident involve alterations in the endothelium as this structure is converted from a passive to an active role in inflammation.’.’ The changes that occur in the endothelium are important in localizing the circulating leukocytes to a restricted area of injury where the infectious process is originating. The actual mechanism(s) that leads to the generation of an “inflamed” or activated endothelium is likely multifactoral. Bacteria-derived products, such as lipopolysaccharide (LPS), can serve as powerful agents for stimulating endothelial cells.* In addition, early response cytokines, including interleukin1 alpha (ILla), interleukin-1 beta (IL-lp), and tumor necrosis factor alpha (TNFa), are also classic mediators that can activate the endothelium.’ Interestingly, LPS can either directly stimulate or indirectly activate endothelial cells by induction of resident, tissue macrophage-derived IL1 or TNF.3,4 Expression of these cell-derived mediators can establish a cytokine network necessary to fully drive the inflammatory respon~e .~ .~ Independent of an exogenous (LPS) or endogenous (IL1 or TNF) signal, activation of the endothelium results in the expression of adhesion molecules on the surface of endothelial cells and leukocytes. The net result of this interaction is the localization of specific leukocyte populations to an area of tissue injury. Elegant studies have demonstrated that the endothelial cell-leukocyte adhesion process is comprised of discrete steps, which include an initial “rolling” event followed by tenacious binding by strong leukocyte-to-endothelial cell interactions. Once tethered to endothelial cells in a local area of tissue injury, the leukocyte must next traffic out of the vessel lumen, move through the basement membrane, and successfully amve at the inflammatory site. Thus, binding of leukocytes to the endothelium is a transient event.

Role of Macrophage Inflammatory Protein (Mip)-Lα in Granulomatous Inflammation

Macrophage inflammatory protein (MIP)-1 is a low MW, LPS-inducible monocyte and neutrophil chemotactic cytokine which may be important in inflammation and pulmonary granuloma formation. The present study examined the kinetic generation, and in vivo relevance of murine MIP- lα utilizing synchronous pulmonary Schistosoma mansoni egg granulomas. Antigenic MIP-lα was measured in 24 hr supernatants from whole granulomas cultured (700/ml) with or without soluble egg antigen (SEA) utilizing an ELISA developed in our laboratory. Intact primary granulomas isolated at various times of development from normal mice showed low backgroud levels of MIP- lα production (<1 ng/ml), however, when challenged with antigen demonstrated significant production of MIP-lα beginning at day 8 (2.6 ng/ml) and peaking at day 16 (16.8 ng/ml). Intact pulmonary granulomas isolated from acutely infected mice demonstrated high backgroud levels of MIP-lα production (peaking at day 2; 7.8 ng/ml). Likewise, background levels from chronic infection granulomas (day 2; 5.6 ng/ml) were similar to acute granulomas. Antigen stimulation increased expression of MIP- lα at all time points with granulomas from acutely infected (peaking at day 2; 16 ± 4.6 ng/ml) but not chronically infected (peaking at day 8; 3.45 ± 1.3 ng/ml) mice. Treatment of mice with polyclonal rabbit anti-mouse MIP-lα (6,125 ± 310 um2) abrogated 8 day primary pulmonary granuloma formation when compared to normal serum control group (12,704 ± 1154 um2). Anti-MIP-lα sera also decreased granuloma formation in lungs of acutely (4-day; 27,897 ± 2400 um2), but not chronically (4-day; 15257 + 1058 um2) infected mice compared to normal serum treated groups (36,010 ± 2507 and 17319 ± 1016 um2, respectively).