Antibody to beta3 integrin inhibits osteoclast-mediated bone resorption in the thyroparathyroidectomized rat.

The cell surface integrin, alphaVbeta3, is important for the attachment of osteoclasts to bone matrix and the subsequent resorption of bone. The present study was designed to determine the effects of F11, a monoclonal antibody to the rat beta3 subunit, on calcium mobilization in a rat model of bone resorption. Male Sprague Dawley rats became hypocalcemic within 18 h after thyroparathyroidectomy. Synthetic PTH-related protein (PTHrP(1-34)) administered to control rats caused serum calcium to return to normal. Anti-beta3 treatment of rats after thyroparathyroidectomy inhibited the calcemic response to PTHrP by 65%. Circulating F11 was biologically active as demonstrated by osteoclast retraction and by the inhibition of adenosine diphosphate-induced platelet aggregation via inhibition of the platelet integrin alphaIIbbeta3 in ex vivo assays. F11 antibody was localized by immunohistological staining to osteoclasts in long bones, suggesting that the mechanism of action of the antibody was via a direct effect upon osteoclasts. Echistatin and calcitonin also inhibited calcemic responses to PTHrP in this in vivo model, whereas an isotype-matched, control antibody was ineffective. These studies provide the first direct evidence in vivo that osteoclast-mediated bone resorption is regulated via beta3 integrin.

Cytokine-induced neutrophil chemoattractant mediates neutrophil influx in immune complex glomerulonephritis in rat.

Chemokines are a family of cytokines whose participation in inflammation in vivo remains to be established. Using the rat model of anti-glomerular basement membrane (GBM) nephritis, we found that mRNA for the chemokine CINC (cytokine-induced neutrophil chemoattractant) was induced in the kidney, and the corresponding protein was elaborated by isolated inflamed glomeruli. Production of CINC by glomeruli was unaffected by complement- or leukocyte-depletion prior to disease induction. Cytokines which induce CINC expression in renal cells (TNF-alpha and IL-1 beta) were also expressed in the kidney during glomerular inflammation. TNF-alpha production, unlike CINC, was complement and leukocyte dependent. In vivo administration of anti-CINC, but not anti-human IL-8, IgG selectively attenuated the influx of PMNs into the glomerulus and commensurately diminished proteinuria. The participation of CINC was not tissue-specific: anti-CINC IgG also diminished the influx of PMNs in dermal immune complex inflammation. In sum, we propose that glomerular immune complex deposition/complement activation leads to cytokine production which results in CINC expression by endogenous glomerular cells. The CINC produced plays a contributory role in the influx of PMNs into the glomerulus in the context of the activation of other inflammatory mediators. These results suggest a potential role for CINC homologues, IL-8 and the GRO family of chemokines, in human immune complex-mediated disease.

Investigation of possible autocrine functions for rat GRO/CINC (cytokine-induced neutrophil chemoattractant).

Rat cytokine-induced neutrophil chemoattractant (CINC) is an eight kilodalton polypeptide originally purified from media conditioned by interleukin-1 beta stimulated 52E, an epithelioid clone derived from normal rat kidney (NRK) cells. Using a fibroblastic clone of the NRK cells, 49F, we found expression of the CINC gene to be induced by either serum or cytokines in growth-arrested cultures within 1 hour of stimulation. There was no observable CINC expression in exponentially growing cells in the absence of cytokine stimulation. CINC protein had no significant effect on 3H-thymidine incorporation or growth rate of NRK49F. We have observed that CINC is constitutively produced by some transformed NRK cells, clone RC20, suggesting an association with the expression of a transformed phenotype. Unlike the parent 49F, RC20 cells are capable of growth in soft agar and serum-free media and form highly metastatic tumors in nude mice. We have examined the possible autocrine functions of CINC and its possible links to the expression of the transformed phenotype by these cells. The use of a blocking CINC polyclonal antibody demonstrated that CINC did not function as an autocrine growth factor for RC20. Though CINC is a potent chemoattractant for neutrophils, it did not induce migration of either RC20 or 49F cells. CINC only moderately promoted adhesion of RC20 cells when used as a matrix protein. These data do not support the hypothesis that production of CINC by the RC20 cells provides an obvious advantage for the transformed cells constitutively producing it.

High-level expression of cytokine-induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: purification and production of blocking antibodies.

Significant levels of cytokine-induced neutrophil chemoattractant (CINC) were found in serum-free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoattractant for rat neutrophils (EC-50 0.5 nM). A fusion protein of glutathione-S-transferase and CINC (GST-CINC) was produced in E. coli. Anti-CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST-CINC affinity chromatography. Both goat and rabbit anti-CINC antibody preparations at 4 micrograms/mL (an 11-fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay. These antibodies have been used to develop a sensitive immunoassay for CINC. The availability of large amounts of affinity-purified blocking anti-CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells.