Regulation of interleukin-8 binding and function by heparin and alpha2-macroglobulin.

BACKGROUND: Increased expression of interleukin-8 (IL-8), a potent neutrophil chemoattractant, is associated with a number of inflammatory diseases. Interleukin-8 binds to the glycosaminoglycan (GAG) heparin and the protease inhibitor alpha2-macroglobulin, molecules which regulate the function of a number of cytokines. Heparan sulphate was previously shown to enhance neutrophil chemotactic responses to IL-8. OBJECTIVE: The purpose of this study was to investigate the effect of heparin, heparan sulphate and alpha2-macroglobulin on IL-8 binding to neutrophils and subsequent functional effects in vitro. METHODS: The binding of 125I-IL-8 to normal neutrophils at 4 degrees C was studied and the IL-8 induced neutrophil chemotactic response was investigated using micro-Boyden chambers. Complexation of IL-8 with alpha2-macroglobulin was confirmed using gel filtration chromatography. RESULTS: Heparin, but not heparan sulphate, inhibited the binding of 125I-IL-8 to neutrophils (IC50=26 microg/mL) and IL-8 induced neutrophil chemotactic responses (IC50=4 microg/mL). The specific inhibitory effect of heparin was apparently due to an interaction with IL-8 which was charge-dependent, since dextran sulphate had a greater inhibitory effect on chemotactic responses (IC50=2 microg/mL) and FITC-heparin did not bind to neutrophils. The heparin-induced inhibition of IL-8 binding and chemotactic responses was reversed in a dose-dependent manner in the presence of alpha2-macroglobulin. The binding of 125I-IL-8 to neutrophils in the presence of alpha2-macroglobulin appears to be, in part, through the specific IL-8 receptor. CONCLUSION: These results point to an anti-inflammatory role for heparin and a novel, potentially, pro-inflammatory role for alpha2-macroglobulin which together indicate the importance of cytokine-binding macromolecules in determining net cytokine function.

The local lymph node assay: an interlaboratory evaluation of interleukin 6 (IL-6) production by draining lymph node cells.

The murine local lymph node assay is a predictive method for the identification of skin-sensitizing chemicals in which activity is measured as a function of proliferative activity induced in lymph nodes draining the site of exposure. In the present study, the induction by topically applied chemicals of draining lymph node cell (LNC) production of the cytokine interleukin 6 (IL-6) has been evaluated as an alternative endpoint for the local lymph node assay. In addition, results derived from studies of IL-6 production by LNC performed independently in two separate collaborating laboratories have been compared. Of the nine skin sensitizing chemicals examined, six provoked detectable levels (> 150 pg ml-1) of IL-6 production by draining LNCs (as measured by enzyme-linked immunosorbent assay) following exposure of mice to at least one test concentration of the material in both of the laboratories. Three other sensitizing chemicals failed to induce measurable IL-6 production at any test concentration in either one or both of the participating laboratories. Both of the non-sensitizing chemicals evaluated (sodium lauryl sulphate and methyl salicylate) also failed to result in detectable IL-6 synthesis. There was a high level of agreement between the two laboratories. The rank order of chemicals with respect to IL-6 production by LNCs was identical in both cases, as was the dose-response relationship observed with each test material. These data reveal that, although inducible IL-6 production by draining LNCs provides a robust approach to the measurement of strong sensitizing activity, as performed here the method is of insufficient sensitivity for the routine identification of skin allergens.

Inducible interleukin-4-secreting cells provoked in mice during chemical sensitization.

It has been demonstrated previously that chemical contact and respiratory allergens differ with respect to the quality of immune responses they will provoke in mice. Trimellitic anhydride (TMA), a human respiratory allergen, induces in mice responses consistent with the preferential activation of Th2-type cells, resulting in the production of IgE anti-hapten antibody and an increase in the serum concentration of IgE. In contrast, oxazolone (OX), a potent contact allergen considered not to cause respiratory hypersensitivity, induces instead Th1-type responses in mice characterized by vigorous IgG2a antibody production and a failure to elicit IgE. In the present study we have extended these investigations and have examined the capacity of these chemicals to stimulate inducible interleukin-4 (IL-4) production by draining lymph node cells (LNC). IL-4 was measured in the supernatants of draining LNC cultured for various periods in the presence or absence of concanavalin A (Con A). Following primary topical exposure to the chemical allergens, Con A-stimulated LNC from OX-treated mice secreted significantly more IL-4 than did LNC from mice exposed to trimellitic anhydride (TMA). A different pattern of IL-4 secretion was observed following culture with Con A of LNC prepared from lymph nodes draining the sites of secondary exposure to these chemicals. In this case significantly higher concentrations of IL-4 were produced by TMA-treated mice. Detectable levels of IL-4 (> 300 pg/ml) were not found following culture of draining LNC from sensitized mice in the absence of Con A or following culture of LNC from naive mice with or without Con A. These data demonstrate that chemical allergens of different types stimulate discrete and changing patterns of inducible IL-4 synthesis consistent with the selective activation of Th-cell subpopulations.

Interleukin 6 production in vitro: An alternative read-out for the local lymph node assay.

The murine local lymph node assay has been developed as an alternative method for the identification of contact allergens. In contrast to guinea pig tests, which rely on visual assessment of challenge-induced dermal reactions, the local lymph node assay measures events occurring during the induction of skin sensitization. Contact allergic potential is measured as a function of hyperplastic responses in draining lymph nodes following systemic administration of [(3)H]thymidine. We have now examined whether the production in vitro of interleukin 6 (IL-6) by draining lymph node cells isolated from sensitized mice provides an alternative endpoint for the local lymph node assay. In comparative experiments, the production of IL-6 by lymph node cells in culture correlated closely with proliferative responses in vitro. Only chemicals known to cause contact sensitization elicited measurable (> 150 pg/ml) IL-6 production; non-sensitizing chemicals, including skin irritants, did not. Experience to date suggests that IL-6 production may provide a useful alternative read-out for the identification of chemicals which have a significant skin-sensitizing potential.