Cytokine-induced neutrophil chemoattractant mediates neutrophilic alveolitis in rats: association with nuclear factor kappa B activation.

Cytokine-induced neutrophil chemoattractant (CINC) is a rat cytokine with structural and functional homology to human interleukin-8 (IL-8) and melanoma growth-stimulatory activity (MGSA/gro). We investigated the relationship between CINC and the production of chemotactic activity for neutrophils by rat alveolar macrophages after in vitro and in vivo treatment with endotoxin. After in vitro treatment with endotoxin, the chemotactic bioactivity produced by alveolar macrophages increased in a time- and dose-dependent manner. This increase in chemotactic activity was closely associated with increased levels of steady-state CINC mRNA. About 50% of the chemotactic activity was blocked by treatment with neutralizing concentrations of anti-CINC antibodies. We then evaluated the role of CINC in vivo in the development of neutrophilic alveolitis in rats, which results from a single intraperitoneal injection of endotoxin. In this model, peak numbers of neutrophils are recovered in lung lavage fluid 24 h after endotoxin injection. Steady-state CINC mRNA levels in the lung peaked 2 h after endotoxin injection. Many cytokines whose transcription is induced during sepsis, including IL-8 and MGSA/gro, are thought to be transcriptionally regulated by nuclear factor kappa B (NF-kappa B). The CINC gene contains a binding site in the promoter region for NF-kB. Therefore, we sought to determine whether NF-kappa B binding to the CINC NK-kappa B motif was increased in nuclear extracts from rat lung lavage cells after exposure to endotoxin using gel mobility shift assays. Increased nuclear NF-kappa B binding activity was detected 2.5 h after in vivo treatment with endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cytokine-induced neutrophil chemoattractant (CINC) mRNA production in cultured rat cells.

Rat cytokine-induced neutrophil chemoattractant (CINC) is an 8-kD polypeptide originally purified from media conditioned by interleukin-1 beta (IL-1 beta)-stimulated 52E, an epithelioid clone derived from the normal rat kidney (NRK) cell line. Using a fibroblastic clone of NRK cells, 49F, we found that lipopolysaccharide (LPS), IL-1 beta, and tumor necrosis factor-alpha (TNF-alpha) each induce synthesis of CINC mRNA and CINC, although in qualitatively and quantitatively different patterns. Through deadenylation experiments and by probing with oligonucleotides, we discovered that the smaller of the two major CINC transcripts appears to arise from the larger as a result of poly(A) tail removal and/or 3' cleavage.

Expression of active human GRO beta and GRO gamma neutrophil chemotactic proteins in E. coli.

Human GRO alpha, GRO beta, and GRO gamma are neutrophil chemoattractants structurally related to IL-8 and compete with IL-8 for binding to IL-8 receptors on neutrophils. These proteins are part of a large superfamily of chemotactic cytokines, the "chemokines," members of which share striking structural similarities. We have expressed GRO cDNA's in Escherichia coli as fusions to the MalE gene product, maltose-binding protein (MBP), in a way that allows separation of GRO and MBP moieties by factor Xa cleavage. GRO beta and GRO gamma expressed in bacteria were active in in vitro chemotaxis assays and were as effective as IL-8 in inducing chemotactic migration of neutrophils. Recombinant GRO beta was chemotactic rather than chemokinetic when tested by checkerboard analysis while GRO gamma showed evidence of chemokinetic as well as chemotactic activity. The activities of GRO beta and GRO gamma were not species-specific as both proteins were active on rat as well as human neutrophils and were inhibitable by antibodies raised against CINC, the rat GRO homolog. These data indicate that the MBP fusion protein expression system provides a rapid and simple method for obtaining large quantities of members of the chemokine protein family for biological uses.

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.

Rat CINC (cytokine-induced neutrophil chemoattractant) is the homolog of the human GRO proteins but is encoded by a single gene.

Human GRO and rat CINC proteins are members of the pro-inflammatory "chemokine" superfamily of chemotactic cytokines which includes IL-8. We have used Northern and Southern blot analyses to compare the expression of CINC-related and GRO transcripts in human cells and to compare the hybridization patterns of CINC and GRO probes in human and rat genomic DNA. Our data indicate that rat CINC is encoded by a single gene unlike GRO-alpha, -beta and -gamma which are encoded by three distinct genes and that they are the nearest homologs to each other from their respective species.

Specific binding of vascular permeability factor to endothelial cells.

Vascular permeability factor (VPF), also known as vascular endothelial cell growth factor, has recently been purified from guinea pig, human, and bovine sources. We show that various fetal or adult endothelial cell strains originating from either capillary or large vessels possess specific high affinity and saturable binding sites for guinea pig tumor-derived [125I]VPF. Two classes of sites with KDs of approximately 10 pM and 1 nM were detected for all endothelial cell types examined. Guinea pig [125I]VPF binding to endothelial cells was inhibited by human VPF (ID50 = 0.8 ng/ml) and by suramin (ID50 = 75 micrograms/ml) but not by heparin. Cross-linking experiments revealed specific [125I]VPF-receptor complexes of two types. Most of the complexes migrated very slowing in SDS-PAGE, indicating that they were of very high molecular weight and probably highly cross-linked. A portion of the molecules migrated as 270 kDa complexes, indicating that the molecular weight of the endothelial cell VPF receptor is about 230 kDa.

An improved method of purification of transforming growth factor, type beta from platelets.

Platelets contain high concentrations of TGF-beta and for this reason have been the primary source of materials for its purification. The yield from acid-ethanol extracts of platelets has been reported to be 0.5 microgram/unit of platelets. We have developed a method which yields approximately 2 micrograms/unit of platelets from acid-ethanol extracts. The use of an ion-exchange column followed by RPLC results in a homogeneous product in less time and with fewer manipulations than the previously published methods. A tritiated thymidine incorporation assay using the CC164 cell line is described for the quantitation of TGF-beta.

Antibodies to the epidermal growth factor receptor block the biological activities of sarcoma growth factor.

The role of the epidermal growth factor (EGF) receptor system in mediating the biological activities of sarcoma growth factor (SGF) has been assessed by using specific anti-EGF receptor antibodies. There are two classes of anti-EGF receptor antibodies, those that block binding of 125I-labeled EGF (125I-EGF) and those that do not block binding but do interact with a portion of the EGF receptor on the surface of intact cells. Antisera of both types have been assayed for their capacity to affect the biological activities of SGF. The antisera that block 125I-EGF binding to its receptor block the induction of DNA synthesis in human fibroblasts by either EGF or SGF but not by other polypeptide mitogens. Titration of the anti-EGF receptor antiserum indicates the presence of one population of antibody that blocks the site of both EGF and SGF action. Antisera to the EGF receptor that block 125I-EGF binding also inhibited the SGF-dependent anchorage-independent growth of normal cells in soft agar. The antisera to the EGF receptor that does not block 125I-EGF binding or EGF activity did not inhibit any of the biological activities of SGF. The results suggest that occupation of the EGF receptor is required for both the mitogenic and colony-forming activity of SGF.

Affinity labeling of a transforming growth factor receptor that does not interact with epidermal growth factor.

Membrane components that interact with epidermal growth factor (EGF) and transforming growth factors (TGFs) have been identified by covalent crosslinking to their respective 125I-labeled ligands. Under appropriate conditions, disuccinimidyl suberate or hydroxysuccinimidyl p-azidobenzoate cross-link receptor-bound 125I-labeled EGF to a 140- to 170-kilodalton (kDal) receptor species in membranes from both A431 human carcinoma cells and normal rat kidney cells. 125I-Labeled sarcoma growth factor (SGF), a TGF from virally transformed mouse 3T3 cells, also can be affinity-crosslinked to the 140- to 170-kDal EGF receptor species in membranes from A431 and rat kidney cells. The labeling of this receptor is inhibited when either excess unlabeled EGF or SGF is present during incubation of membranes with either 125I-labeled EGF or 125I-labeled SGF. In contrast, a second receptor species of 60 kDal is affinity-labeled with 125I-labeled SGF but not with 125I-labeled EGF in membranes from both A431 and rat kidney cells. SGF and a TGF from virally transformed rat embryo cells inhibit the labeling of the 60-kDal species when present in excess during incubation of membranes with 125I-labeled SGF, whereas EGF is completely ineffective in inhibiting the labeling of this receptor. The data suggest that a specific 60-kDal receptor that displays high affinity for TGFs but not for EGF may mediate induction of the transformed phenotype. In addition, SGF and other TGFs interact with the 140- to 170-kDal EGF receptor that appears to mediate normal cell growth effects.

In vitro production of immunosuppressive factors by murine sarcoma virus-transformed mouse fibroblasts.

Murine sarcoma virus-transformed mouse fibroblasts produce potent immunosuppressive factors (ISF) in vitro. The partially purified ISF inhibited thymocyte proliferation induced by concanavalin A or phytohemagglutinin plus lymphocyte activating factor (Interleukin 1), lipopolysaccharide-induced spleen cell proliferation, the in vitro splenic anti-sheep erythrocyte plaque-forming cell response, and the generation of alloantigen-specific cytotoxic T cells. The effect of ISF on thymocyte proliferation was not readily reversible and required only a 4-hr exposure of the thymocytes to ISF to inhibit cell proliferation. Although ISF shares several biochemical properties with a murine sarcoma virus-transformed cell-derived sarcoma growth factor (e.g., acetic acid solubility and sensitivity to dithiothreitol), the two factors could be resolved by gel filtration on Bio-Gel P-60. Two peaks of ISF activity were found with apparent molecular weights of 12,000 and 8000. The results described here support the hypothesis that at least some of the ISF obtained from the serum of tumor-bearing hosts may be released by the tumor cells themselves. In view of the potent in vitro activity of the murine sarcoma virus-transformed fibroblast-derived ISF, it is quite possible that ISF-like molecules may play a role in subverting in vivo tumor rejection processes involving the immune system.