Stephen W. Chensue

Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung.

Cellular constituents of the alveolar-capillary wall may be key participants in the recruitment of polymorphonuclear leukocytes to the lung through the generation of the novel neutrophil chemotactic peptide interleukin-8 (IL-8). This interaction appears to occur via the ability of human alveolar macrophage (AM)-derived monokines, tumor necrosis factor (TNF), and interleukin-1 (IL-1) to induce gene expression of IL-8 from pulmonary type II-like epithelial cells (A549). Northern blot analysis demonstrated that steady-state IL-8 mRNA expression, by either TNF- or IL-1 beta-treated A549 cells, occurred in both a dose- and time-dependent fashion. Similarly, extracellular antigenic IL-8, as assessed by specific ELISA, was expressed from TNF- or IL-1 beta-stimulated epithelial cells in a time-dependent fashion with maximal IL-8 antigen detected at 24 h poststimulation. Immunohistochemical staining utilizing rabbit anti-human IL-8 antibody identified immunoreactive, cell-associated IL-8 antigen as early as 8 h post-TNF or IL-1 beta stimulation. A549-generated neutrophil chemotactic bioactivity paralleled IL-8 steady-state mRNA levels. Signal specificity was demonstrated in this system as IL-8 mRNA or protein expression by lipopolysaccharide (LPS)-treated A549 cells was not different from unstimulated cells. Although LPS did not serve as a direct stimulus for the production of IL-8 by type II-like epithelial cells, the condition media from LPS-challenged AM induced a significant expression of IL-8 mRNA by the A549 cells. 24-h conditioned media from LPS-treated cells was as potent as either IL-1 beta or TNF in generating steady-state IL-8 mRNA by A549 cells. Preincubation of LPS-treated AM-conditioned media with anti-human TNF or IL-1 beta neutralizing antibodies resulted in significant abrogation of IL-8 gene expression by A549 pulmonary epithelial cells. These findings demonstrate potential cell-to-cell communication circuits that may be important between AMs and pulmonary epithelial cells during the recruitment phase of acute lung inflammation.

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.

Role of Lipoxygenase Products in Murine Pulmonary Granuloma Formation

Various arachidonic acid (AA) metabolites are known to regulate immune cell function(s) and dictate the progression of both acute and chronic inflammatory reactions. Using a model of Schistosoma mansoni egg-induced hypersensitivity granulomas, we have delineated the in vivo effects of inhibitors of cyclooxygenase (CO) and lipoxygenase (LO) pathways on granuloma development and granuloma macrophage I-region-associated (Ia) antigen expression. In addition, by high performance liquid chromatography (HPLC) we have profiled the metabolism of AA by macrophages that are isolated from granulomatous foci, and have biochemically characterized the in vitro specificity and activity of selected CO and LO inhibitors. The development of hypersensitivity-type pulmonary granulomas in mice was dramatically suppressed by inhibitors with anti-LO activity (nordihydroguairetic acid (NDGA), nafazatrom, and BW755c) in a dose-dependent manner, while indomethacin, which is primarily CO-selective, had no significant effect. Furthermore, NDGA and nafazatrom profoundly arrested the normal progression of preformed granulomatous lesions. The inhibitors of the LO pathway also suppressed the in vivo kinetics of Ia antigen expression by granuloma macrophages. In contrast, indomethacin augmented Ia-antigen expression. The major AA metabolites that were synthesized by the granuloma macrophages were shown to be leukotriene C4 and mono-hydroxyeicosatetraenoic acids. HPLC analysis of AA metabolites from granuloma macrophages that were treated with the various inhibitors confirmed that indomethacin was most CO-selective and NDGA most LO-selective. Nafazatrom and BW755c inhibited AA metabolism by both pathways. Notably, high concentrations of the compounds (5 X 10(-5) M) tended to suppress all products. Our results suggest that LO products may be important in the generation and maintenance of immune granulomatous inflammatory responses.

Biologic and immunohistochemical analysis of macrophage interleukin- 1 alpha, - 1 beta, and tumor necrosis factor production during the peritoneal exudative response.

The present study examined changes in lipopolysaccharide (LPS)‐induced interleukin 1 (IL‐1) and tumor necrosis factor (TNF) production by murine peritoneal macrophages during the chronic exudative response to Freunds complete adjuvant (CFA). Macrophages were isolated by peritoneal lavage and adherence at intervals over a 32 day period following i.p. injection of CFA. Optimal culture conditions for IL‐1 and TNF production were predetermined, and it was found that IL‐1 production was profoundly impaired at densities of above 150 cells/mm2, whereas TNF sythesis was more resistant to density effects. Using optimal conditions, we observed a sequential appearance of monokines. On day 0 there was minimal IL‐1 production and no detectable TNF production. By days 4–7, IL‐1 production reached maximum levels with a steady decline to baseline by day 32. TNF production steadily increased after day 2, reached maximal levels by days 16–20, and then partly declined by day 32. These findings were supported by kinetic analyses at specified days. When related to exudative events, it appeared that maximal IL‐1 was associated with the recruitment stage of the reaction, whereas TNF production was associated with the established exudate. Immunohistochemical analysis revealed that TNF production could be related to the proportion of macrophages with cytoplasmic TNF expression. In contrast, IL‐1α and ‐1β expression was comparable among populations with 85–100% of cells showing cytoplasmic expression 6 hr after LPS stimulus. Whereas cytoplasmic IL‐1α persisted for the 18 hr study period, IL‐1β disappeared from many adjuvant recruited cells. Our findings suggest that monokine production is orchestrated during macrophage recruitment and activation at sites of chronic inflammation.

lnterleukin-6 Expression in Immunologically Elicited Murine Macrophages

We report the expression of both interleukin-6 (IL6) messenger RNA and biological activity in complete Freunds adjuvant-elicited peritoneal macrophages (CFA-M phi). IL6 mRNA expression peaked between 4 and 8 h of lipopolysaccharide (LPS) stimulation; biological activity was maximal at approximately 18 h of stimulation. LPS-induced IL6 mRNA was inhibited by treatment with cycloheximide (5 micrograms/ml), implicating the participation of a secondary protein mediator in the induction process, or the dependence upon protein synthesis for receptor ligand interactions. Comparison of CFA-M phi with resident peritoneal macrophages suggests that the elicited cell population makes more IL6 in response to LPS than the resident population on a per cell basis.

The Function of Chemokines in Health and Disease

The cascade of events that dictate the normal physiologic processes leading to the initiation, maintenance, and final resolution of inflammation is the result of the host responding to a variety of direct or indirect stimuli. Although these stimuli may represent either infectious agents (viruses, bacteria, and protozoans) or noninfectious processes (trauma, autoimmune disorders, and ischemia/reperfusion injury), they all result in the activation and directed migration of leukocytes into an area of tissue injury. Our current understanding of inflammation suggests that the recruitment of leukocytes from the lumen of a vessel into a localized area of injury depends on an interrelated network of events, which must occur with some fidelity in order for the cells to arrive successfully at a site of inflammation. Although many of the steps involved in leukocyte activation and elicitation have been identified, a complete understanding of these processes, including the subsequent tissue injury, are not entirely known.

The Role of CC Chemokines in Th1- and Th2-Type Pulmonary Inflammation Models

Elucidation of the factors involved in the initiation and persistence of inflammatory diseases within the interstitial space of the lung has garnered great research interest over the past two decades. Interstitial lung diseases are defined as pulmonary inflammatory disorders that lead to excessive tissue injury and progressive fibrosis. Idiopathic fibrosis and end-stage sarcoidosis are two clinically predominant examples (1). Whereas the list of inflammatory mediators present in interstitial lung diseases has grown immensely to include cytokines, growth factors, prostanoids, eicosanoids, and free radicals (2) little is presently known about the manner in which these factors initiate and sustain these complex diseases. An inflammatory response to infection or injury within the lung is typically characterized by the de novo generation of inflammatory mediators by tissue resident immune (i.e., alveolar macrophages and mast cells) and nonimmune (i.e., epithelial) cells. These mediators modulate the function of these cells and also promote the infiltration of lymphocytes and phagocytes, and the concerted efforts of all of these cells facilitate the destruction and/or clearance of the inflammatory stimulus. Once this aim has been achieved, the last stage of the inflammatory response involves the restoration of the tissue to its original state. Clearly, the recruitment and tissue-reparative stages must be closely regulated in the lung because excessive tissue injury or overexuberant tissue healing can significantly disrupt the pulmonary architecture and even lead to respiratory failure (3). In prolonged inflammatory diseases of the lung, clinical and laboratory evidence suggest that the inability of the lung to contain the inflammatory process and/or repair itself correctly is related to the persistence of one or several inflammatory signals (4).