Ichiro Naito

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

A group of rat monoclonal antibodies recognizing the six different α 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 α 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 α 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.

Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H+-ATPase.

To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H+-ATPase and analysis by confocal images showed that ClC-5 and H+-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H+ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.

Differential expression of type IV collagen isoforms, α5(IV) and α6(IV) chains, in basement membranes surrounding smooth muscle cells

Abstract Smooth muscle is composed of cigar-shaped, non-striated cells, each of which is encapsulated by a basement membrane and forms the contractile portion of tubular organs such as the gastrointestinal tract, pulmonary tract, genitourinary tract, and vasculature, in which slow and sustained contractions are needed. We examined basement membranes produced by smooth muscle cells and, using α(IV) chain-specific monoclonal antibodies, analyzed type IV collagens in these organs. Detailed distribution analysis of the α chains in normal and Alport cases by use of specific antibodies indicated that there are at least three molecular forms of type IV collagen, [α1(IV)]2α2(IV), α3(IV)α4(IV)α5(IV), and α5(IV)/α6(IV). Smooth muscle cells in the urinary bladder and uterus were enclosed by basement membranes composed of α1, α2, α5, and α6 chains. The same α chains were present around smooth muscle cells in the muscular layer of the fundus of the stomach, whereas those in the antrum and further distal side of the gastrointestinal tract expressed mostly α1 and α2 chains. In addition, immunostaining analysis of the vasculature also showed that most of the smooth muscle cells were positive for α1 and α2 chains; however, α5 and α6 chains were also expressed by smooth muscle cells in the aorta and some arteries where blood pressure changes significantly. These results suggest that the smooth muscle cells enclosed by α5/α6-containing basement membranes might have some particular function related to mechanical stress or tensile strength during the characteristic contractile activity of tubular organs.

Decreased expression of B7 costimulatory molecules and major histocompatibility complex class-I in human hepatocellular carcinoma

Background and Aim:  We analyzed the expression of antigen‐processing and antigen‐presenting molecules in surgically resected fresh samples of human hepatocellular carcinoma (HCC) tissue to elucidate a mechanism of immune escape. We also examined the expression of interleukin (IL)‐10 protein, which might act to downregulate expression of antigen‐processing and antigen‐presenting molecules.

Irradiation Prolongs Survival of Alport Mice

Alport syndrome is a hereditary nephropathy that results in irreversible, progressive renal failure. Recent reports suggested that bone marrow transplantation (BMT) has a beneficial, short-term effect on renal injury in Alport (Col4a3(-/-)) mice, but its long-term effects, especially with regard to survival, are unknown. In this study, Alport mice received a transplant of either wild-type or Col4a3(-/-) bone marrow cells. Surprising, laboratory evaluations and renal histology demonstrated similar findings in both transplanted groups. Transplanted cells accounted for >10% of glomerular cells at 8 wk, but type IV collagen alpha3 chains were not detected in glomerular basement membranes of either group by immunofluorescence or Western blot analysis, although Col4a3 mRNA in the kidney could be amplified by reverse transcription-PCR in knockout mice that received a transplant of wild-type bone marrow. Both transplanted groups, however, survived approximately 1.5 times longer than untreated knockout mice (log rank P < 0.05). These data suggested that irradiation, which preceded BMT, may have conferred a survival benefit; therefore, the survival time of knockout mice was assessed after sublethal irradiation (3, 6, and 7 Gy) without subsequent BMT. A strong positive correlation between irradiation dosage and survival time was identified (P < 0.0001). In conclusion, the improved survival observed in Alport mice that received a transplant of wild-type bone marrow might be primarily attributed to as-yet-unidentified effects of irradiation.

The Inner Ear of Dogs with X-Linked Nephritis Provides Clues to the Pathogenesis of Hearing Loss in X-Linked Alport Syndrome

Alport syndrome is an inherited disorder of type IV collagen with progressive nephropathy, ocular abnormalities, and high-tone sensorineural deafness. In X-linked Alport syndrome, mutations in the COL4A5 gene encoding the alpha5 chain of type IV collagen lead to loss of the alpha3/alpha4/alpha5 network and increased susceptibility of the glomerular basement membrane to long-term damage. The molecular defects that underlie the otopathology in this disease remain poorly understood. We used a canine model of X-linked Alport syndrome to determine the expression of type IV collagen alpha-chains in the inner ear. By 1 month in normal adult dogs, the alpha3, alpha4, and alpha5 chains were co-expressed in a thin continuous line extending along the basilar membrane and the internal and external sulci, with the strongest expression along the lateral aspect of the spiral ligament in the basal turn of the cochlea. Affected dogs showed complete absence of the alpha3/alpha4/alpha5 network. The lateral aspect of the spiral ligament is populated by tension fibroblasts that express alpha-smooth muscle actin and nonmuscle myosin and are postulated to generate radial tension on the basilar membrane via the extracellular matrix for reception of high frequency sound. We propose that in Alport syndrome, the loss of the alpha3/alpha4/alpha5 network eventually weakens the interaction of these cells with their extracellular matrix, resulting in reduced tension on the basilar membrane and the inability to respond to high frequency sounds.

Differential distribution of basement membrane type IV collagen α1 (IV), α2 (IV), α5 (IV) and α6 (IV) chains in colorectal epithelial tumors

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen α chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, α1/α2(IV) and α5/α6(IV) chains were stained in all epithelial BM. However, α3/α4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for α1/α2(IV) and α5/α6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both α1/α2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of α5/α6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of α5/α6(IV), partly α3/α4(IV) chains, in addition to the classic network of α1/α2(IV) chains. The differential immunohistochemical localization of the type IV collagen α5/α6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.

Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen

SummaryThe nephritogenic antigen that induces antiglomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in rats was isolated from collagenase-solubilized bovine renal basement membranes. Purification was achieved using antibody-coupled affinity columns which were originally used for the purification of trypsin-solubilized nephritogenic antigen (Sado et al. 1984a). The nephritogenic antigen was a heteropolymer composed of P2 (Mr 28 kDa) and P3 (Mr 30 kDa) polypeptides as monomers and their dimers in sodium-dodecyl-sulfate (SDS) polyacryl amide gel electrophoresis. The P3 polypeptide was considered to be the nephritogenic epitope, since a fraction composed of the P2 polypeptide alone was not nephritogenic.The properties of the nephritogenic epitope were the same as those of the Goodpasture epitope (M2*), which is a noncollagenous domain of the α3 chain of type IV collagen (Butkowski et al. 1985; Saus et al. 1988), indicating that the nephritogenic antigen is the same as the Goodpasture antigen.

Transfer of the α5(IV) Collagen Chain Gene to Smooth Muscle Restores in Vivo Expression of the α6(IV) Collagen Chain in a Canine Model of Alport Syndrome

X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the α5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant α5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine α5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the α1(IV) and α2(IV), but not the α3(IV) to α6(IV) collagen chains. By Northern blotting, an α5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the α1(IV) and α2(IV) chains but the recombinant α5(IV) chain was not incorporated. These findings are consistent with the concept that the α5(IV) chain requires one or more of the α3(IV), α4(IV), or α6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the α5(IV) transgene was injected into bladder smooth muscle that lacks both the α5(IV) and α6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the α5(IV) and α6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant α5(IV) chain is capable of restoring expression of a second α(IV) chain that requires the presence of the α5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome.

Distribution of collagen type IV α1–6 chains in human normal colorectum and colorectal cancer demonstrated by immunofluorescence staining using chain‐specific epitope‐defined monoclonal antibodies

Background and Aim: Loss of basement membrane (BM) components, such as type IV collagen, has been demonstrated in colorectal cancer, but the fine diversity of the assembly of α (IV) chains, the composition of type IV collagen, and alterations in the collagen have not been fully analyzed. Here, we defined immunohistochemically the expression of α1–6 (IV) chains in colorectal cancer tissues and adjacent normal mucosa by the use of chain‐specific monoclonal antibodies.