Clinical Investigation

Kidney International (1991) 39, 1240–1248; doi:10.1038/ki.1991.157

Cytokine- and LPS-induced synthesis of interleukin-8 from human mesangial cells

David J Kusner1, Ellen L Luebbers1, Robert J Nowinski1, Martha Konieczkowski1, Charles H King1 and John R Sedor1

1Department of Medicine, School of Medicine, Case Western Reserve University, and Divisions of Geographic Medicine and Nephrology, University Hospitals, Cleveland, Ohio, USA

Correspondence: John R Sedor MD, Division of Nephrology, Department of Medicine, University Hospitals, 2074 Abington Road, Cleveland, Ohio 44106, USA.

Received 31 October 1990; Revised 22 January 1991; Accepted 23 January 1991.

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Abstract

Cytokine- and LPS-induced synthesis of interleukin-8 from human mesangial cells. The cytokine neutrophil-activating peptide-1/interleukin-8 (NAP/IL-8) activates neutrophils (PMN) and elicits selective diapedesis of PMN into the extracellular space. The glomerular mesangial cell (MC) is a specialized pericyte that controls glomerular filtration and synthesizes and responds to a variety of cytokines. Because of its location within the glomerulus, the MC is in a pivotal position to orchestrate events underlying immune injury. Since immune-injured glomeruli have been shown to produce NAP/IL-8 activity in vitro, we assessed whether lipopolysaccharide (LPS)- or cytokine-activated MC might be a source of this activity. Pure human MC, devoid of monocyte/ macrophage and fibroblast contamination, were grown by expiant from collagenase-treated glomeruli. Human recombinant interleukin-1 alpha (IL-1alpha, 20 ng/ml), IL-1beta (50 ng/ml), tumor necrosis factoralpha (TNF, 100 ng/ml) and lipopolysaccharide (LPS, 10 microg/ml) stimulated release of a neutrophil chemotactic factor from cultured MC. Both concentrated (fivefold) and unconcentrated MC supernatants stimulated directed neutrophil migration under agarose at a level similar to that of the bacterial chemotactic factor, FMLP. In contrast, unstimulated MC-conditioned media and IL-1alpha, IL-1beta, TNF and LPS in medium alone did not directly induce PMN migration. Molecular sizing studies using sequential membrane ultrafiltration identified significant TNF-stimulated, MC-derived chemotactic activity in the 3000 to 10000 kD fraction. An anti-NAP/IL-8 monoclonal antibody, 46E5, significantly inhibited PMN chemotaxis stimulated by TNF-stimulated, MC-conditioned media in a dose-dependent manner. S1 nuclease analyses of a total RNA extracted from TNF-stimulated MC showed a dose- and time-dependent increase in NAP/IL-8 mRNA transcripts and demonstrated that a chemotaxin, produced by cytokine- and LPS-stimulated MC, is highly homologous to NAP/IL-8. Northern analysis of TNF-stimulated MC RNA identified NAP-l/IL-8 transcripts in MC which comigrated with the 1.8 kb message in LPS-stimulated human monocytes. Using [35S]la-belled oligonucleotide probes, NAP/IL-8 mRNA transcripts were demonstrated in the cytoplasm of IL-1 and TNF-stimulated, but not control, mesangial cells. Taken together, these data demonstrate the human MC synthesize and release NAP/IL-8, a potent chemotactic peptide for PMN that may contribute to the structural and functional derangements which occur in glomerular inflammation.

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