We have shown previously that the 5' ends of the genes for the ca5(IV) and ca6(IV) collagen chains lie head-to-head on

We have been interested in identifying proinflammatory molecules which might play a role in attracting monocytes and T cells to the kidney. Some of the new intercrines are potential candidates. In this report we have isolated cDNA encoding murine Rantes (MuRantes) from renal tubular epithelium (MCT cells). MuRantes is a 91 amino acid member of the -C-C- or intercrine beta subgroup of the Scy superfamily. The amino acid sequence for mature MuRantes was deduced from its coding cDNA and was found to be 90% homologous to its mature human counterpart (HuRantes). MCT epithelium expresses a single mRNA transcript for MuRantes of approximately 1100 bp. The MuRantes protein could be detected in cell lysates of MCT epithelium by western blotting and in the cytoplasm of MCT cells by immunofluorescence using a polyclonal antibody generated against HuRantes fusion protein. A search protocol using MuRantes-specific primers and cDNA amplification revealed that mRNAs for MuRantes are expressed additionally in syngeneic mesangial cells (MMC cells), whole kidney, liver, and spleen, as well as in nephritogenic antigen-specific CD4+ helper and CD8+ effector T cells. cDNA amplification studies also demonstrated a significant elevation in mRNA transcripts encoding MuRantes in response to the stimulation of MCT epithelium with TNF alpha and IL-1 alpha in culture, but not with TGF beta, gamma IFN, or IL-6. Our findings indicate that proximal tubular epithelium is an authentic source of MuRantes, and that transcripts encoding MuRantes are responsive to the modulating influence of paracrine factors having a known role in the development of parenchymal injury.

Fibroblasts in parenchymal organs potentially contribute extracellular matrix to local fibrogenic processes. This contribution, in some circumstances, may be initiated by cytokines disseminated from inflammatory lesions. Different populations of fibroblasts, however, might respond distinctively to this cytokine bath depending on the microenvironment in which they reside. We have begun to explore this issue using syngeneic, low-passage fibroblasts cultured in serum-free media that were derived originally from the dermis (DFBs) and from tubulointerstitium (TFBs) of the kidney. Our findings indicate that, while fibroblasts from each compartment appear similar at the ultrastructural level, there are a variety of functional differences which distinguish their proliferative response, and their collagen secretory response (types I, III, IV, and V) following challenge with various doses of immune-relevant cytokines (TGF beta, EGF, IL-1, IL-2 and gamma IFN) in culture. DFBs, for example, express more surface EGF receptors than do TFBs, and, as a consequence, exhibit a more robust proliferative response to EGF in serum-free media. Unstimulated DFBs also secrete more collagen types I and III than TFBs, while unstimulated TFBs secrete more types IV and V. The expression of these collagens in TFBs was confirmed by Northern blot hybridization. When these sets of fibroblasts were further stimulated by cytokines, some of the cytokines not only differentially effect the secretion of various species of collagens within the same group of cells, but also between cells from populations which are anatomically distinct. DFBs, furthermore, at mid-level doses of cytokine, demonstrated a general trend towards less secretion of all types of collagen (particularly for TGF beta, EGF, and IL-2), while TFBs seemed less repressive. In TFBs the cytokine-induced responses for collagen types I and III tended to be discordant, and for types I and IV EGF inhibited, while TGF beta stimulated the secretory process. These findings speak collectively for the presence of a functional heterogeneity among organ-based populations of syngeneic fibroblasts in normal tissues.

No abstract

The family of type IV collagen comprises six chains numbered ␣1 through ␣6. The ␣3(IV) NC1 domain is the primary target antigen for autoantibodies from patients with anti-basement membrane disease and Goodpasture syndrome. Earlier peptide studies suggested that the last 36 amino acids of the ␣3 NC1 domain probably contains one recognition site for Goodpasture autoantibodies, and an algorithm analysis of secondary structure from a later study predicted a second possible upstream epitope near the triple helix junction. We have used several analytic approaches to evaluate the likelihood of two immunologic epitopes for the Goodpasture antigen. In our first set of studies, peptide antibodies directed against these two putative regions co-inhibited Goodpasture autoantibodies binding to denatured human ␣3(IV) NC1 monomer by nearly 80%, with the helixjunction region of the ␣3 NC1 domain contributing 26% of the binding sites and the C-terminal region contributing the remaining 50%. Second, both of these candidate regions are normally sequestered within the associated ␣3(IV) NC1 hexamer but become more visible for binding by anti-peptide antibodies upon their dissociation, a property that is shared by the Goodpasture autoantibodies. Third, segment deletions of recombinant ␣3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the ␣3(IV) NC1 domain. Since the lysines in that region have been previously advanced as possible contact sites, we created several substitutions within the C-terminal epitope of the ␣3 NC1 domain. Substitution of lysines to alanines revealed lysines 219 and 229 as essential for antibody binding to this distal site; no lysines were present in the NC1 part of the helix-NC1 junction region. Substitutions involving arginine and cysteines to alanines in the same C-terminal region did not produce significant reductions in antibody binding. In summary, our findings characterize two Goodpasture epitopes confined to each end of the ␣3 NC1 domain; one is lysine-dependent, and the other is not. We propose, as a hypothetical model, that these two immunologically privileged regions fold to form an optimal pathogenic structure within the NC1 domain of the ␣3 chain. These sites are subsequently concealed by NC1 hexamer assembly of type IV collagen.