Differential expression of mouse alpha5(IV) and alpha6(IV) collagen genes in epithelial basement membranes.

We first completed the primary structure of the mouse alpha5(IV) and alpha6(IV) chains, from which synthetic peptides were produced and a chain-specific monoclonal antibodies were raised. Expression of collagen IV genes in various basement membranes underlying specific organ epithelia was analyzed by immunohistochemical staining using these monoclonal antibodies and other antibodies from human and bovine sequences. It was possible to predict the presence of the three collagen IV molecules: [alpha1(IV)](2) alpha2(IV), alpha3(IV)alpha4(IV)alpha5(IV), and [alpha5(IV)](2)alpha6(IV). In skin basement membrane two of the three forms, [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV), were detected. The alpha3(IV)alpha4(IV)alpha5(IV) molecule was observed as the major form in glomerulus, alveolus, and choroid plexus, where basement membranes function as filtering units. The molecular form [alpha5(IV)](2)alpha6(IV) was present in basement membranes in tubular organs such as the epididymis, where the tubes need to expand in diameter. Thus, the distribution of the basement membranes with different molecular composition is consistent with tissue-specific function.

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Interleukin-2-dependent augmentation of the anti-TNP antibody production by sodium butyrate in cultured murine splenic B cells.

Previously we found that sodium butyrate (NaBu) markedly enhanced production of the antibody specific for a T-cell-dependent antigen, sheep red blood cells (SRBC) in murine splenocytes (Kishiro, Y., Ueda, K., Fujiwara, M. and Yamamoto, I., Jpn J. Phamacol., 1994 66, 369-376. To gain a better understanding of the target cells for NaBu's action on antibody responses, we have utilized the T-cell-independent antigen, trinitrophenyl-lypopolysaccharide (TNP-LPS) as a stimulant and have examined an effect of NaBu on the anti-TNP antibody production in vitro. NaBu markedly increased the anti-TNP plaque-forming cell (PFC) responses in murine whole splenocytes, but not in murine splenic B cells. Addition of T-cells or the concanavalin A supernatant (CAS) from murine splenocytes to the B cell cultures completely restored the enhancing effect of NaBu. This effect of CAS was totally blocked by an anti-interleukin (IL)-2 antibody and partially by an anti-IL-1 beta or anti-IL-4 antibody. The full enhancing effect of NaBu was also detected when IL-2 was added to the B cell cultures, while IL-2 alone had no stimulatory effect on the control PFC response. IL-1 beta alone significantly stimulated the antibody production and adding NaBu to this IL-1 beta-supplemented culture caused a further increase. Neither IL-4 alone nor NaBu plus IL-4 had any effect on the PFC response. NaBu did not affect the expression of the IL-2 receptor alpha- and beta-chains in B cells stimulated with TNP-LPS. These results suggest that NaBu is an agent that promotes B cell differentiation in vitro in an IL-2-dependent manner.

Epitope-defined monoclonal antibodies against type-IV collagen for diagnosis of Alport's syndrome.

BACKGROUND: Alport's syndrome can be diagnosed by staining the alpha 5 chain of type IV collagen in kidney biopsy specimens with a monoclonal antibody. Because antibodies already established against the alpha 5 chain require denaturation treatment of cryostat sections to expose their epitopes. To save time and effort for staining, a new epitope-defined monoclonal antibody whose epitope is initially exposed on the surface of the molecule was established. METHODS: Two monoclonal antibodies against the triple-helical domains of the type IV collagen alpha 2 and alpha 5 chains were established with synthetic peptides as immunogens by the rat lymph node method. Their epitope were EAIQP at the positions of 675-679 of the alpha 2 chain, and IDVEF at the positions of 251-255 of the alpha 5 chain, respectively. They were purified with synthetic peptide-coupled affinity columns, and then conjugated with Texas red and FITC, respectively. RESULTS: The mixture of fluorochrome-conjugated antibodies was able to detect the distribution of the alpha 2 and alpha 5 chains in the normal and Alport kidney and skin by direct immunofluorescence staining with and without denaturation treatment of the sections. CONCLUSIONS: The direct double immunofluorescence staining of kidney and skin cryostat sections with the fluorochrome-conjugated antibodies is useful, reliable, and convenient for diagnosis of Alport's syndrome.

Purification and characterization of human nephritogenic antigen that induces anti-GBM nephritis in rats.

Human nephritogenic antigen induces anti-glomerular basement membrane antibody glomerulonephritis in rats. This antigen was purified from collagenase-solubilized renal basement membrane by means of gel filtration and affinity chromatography using a rabbit antibody. Western blots of the purified nephritogenic antigen using epitope-defined monoclonal antibodies showed that it contains the NC1 domains of the a1 to a6 chains of type IV collagen. Nephritogenicity was thought to be a feature of the NC1 domains of the a3 to a5 chains, because the a6 chain is not located in the glomerular basement membrane, and because an NC1 fraction consisting of the NC1 domains of the a1 and a2 chains was poorly nephritogenic. Autoantibodies in the sera of patients with Goodpasture's syndrome were detected by ELISA using the purified nephritogenic antigen. These results indicate that the nephritogenic antigen contains the Goodpasture antigen, defined as the antigen reactive with sera from patients with Goodpasture's syndrome.

Nephritogenicity and alpha-chain composition of NC1 fractions of type IV collagen from bovine renal basement membrane.

Nephritogenicity (anti-GBM-nephritis-inducing activity) and alpha-chain composition of globular-do-main (NCI) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different alpha chains of type IV collagen. A purified nephritogenic fraction from renal BM contained alpha 1-alpha 6(IV)NCI, whereas a non-nephritogenic fraction contained only alpha 1-alpha 2(IV)NCI. Renal and pulmonary NCI had strong nephritogenic activity: placental NCI had weak activity. The renal and pulmonary fractions contained alpha 1-alpha 6(IV)NCI, and the placental fraction had a large amount of alpha 1-alpha 2(IV)NCI and a very small amount of alpha 3-alpha 6(IV)NCI. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained alpha 1-alpha 5(IV) chains, but not the alpha 6(IV) chain. The absence of alpha 6(IV) chain in glomerular BM in bovine and in humans indicates that alpha 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of alpha 3-alpha 5(IV)NCI.

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen.

A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.

Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.

A novel method of preparing rat-monoclonal antibody-producing hybridomas by using rat medial iliac lymph node cells.

A novel method of preparing hybridomas producing rat monoclonal antibodies was established. The enlarged medial iliac lymph nodes from rats injected via hind footpads with an emulsion of antigen and Freund's adjuvant were used for cell fusion. Ovalbumin was used as a representative antigen. The incidence of hybridomas producing antibody of interest with this method was about 10 times higher than that of hybridomas with the conventional method using mouse or rat spleen cells. The average percentages of hybridomas producing IgG1, IgG2a, IgG2b and IgG2c were 37.1%, 47.0%, 15.9% and 0.0%, respectively. A single injection with antigen was sufficient for immunization in this method.

Butyrate enhances the in vitro anti-SRBC (sheep red blood cell) antibody responses in murine splenocytes.

Butyrate at concentrations of 200-600 microM markedly enhanced the in vitro antibody productions against sheep red blood cells (SRBC) in murine splenocytes. However, other saturated short-chain fatty acids, including acetate, propionate and valeric acid, and 4-carbon compounds such as butanol, acetoacetate and beta- and gamma-hydroxybutyrate had no such effects. The presence of butyrate in the early phase of the cell culture was crucial for enhancement of the response. Butyrate also augmented the antibody production in T-cell-depleted splenocytes supplemented with the culture supernatant of concanavalin A (Con A)-stimulated lymphocytes. Interleukin (IL)-2 secreted from splenocytes in response to SRBC was increased by adding butyrate to the culture, but IL-1 secretion was not affected. On the other hand, Con A or lipopolysaccharide-stimulated proliferation of splenocytes was partly depressed by the addition of butyrate, while Con A-induced IL-2 production was not effected. These findings suggest that butyrate may act on T and B cells to promote their differentiation during the process of antibody production.