Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Evidence for the involvement of carbohydrate moieties in the adhesion of U937 cells to tumor necrosis factor-α (TNFα)-stimulated vascular endotheliumin vitro

Treatment of U937 cells with fructose 1-phosphate (P) and fucoidan dose-dependently inhibited the adhesion of these monocytic cells to TNF-stimulated human umbilical vein eindothelial cells (HUVEC) (IC₅₀=1 mM and 10 g/ml respectively). These carbohydrates (CHO) failed to inhibit U937 adhesion to unstimulated (basal) HUVEC or phorbol 12, 13 dibutyrate (PdBu)-stimulated HUVEC. At 10 mM concentration, both fucose 1-P and lactose 1-P inhibited TNF-stimulated adhesion while the latter also inhibited basal adhesion. Fructose 6-P, fucose, galactose 1-P, glucose 1-P, glucose 6-P, glucuronic acid,-glycerol 1-P, mannose 1-P, mannose 6-P, ribose 1-P and ribose 5-P tested at 10 mM did not inhibit U937 cells adhesion to basal or TNF-stimulated HUVEC. These data suggest that CHO may play an important role in modulating monocytes adhesion to cytokine-induced adhesion molecule(s) on the surface of HUVEC.     

Rho protein inhibition blocks cyclooxygenase-2 expression by proinflammatory mediators in endothelial cells

Rho proteins participate in the regulation of inflammatory gene expression in endothelial cells. We made use of Clostridium difficile toxin B-10643 (TcdB-10463) which inhibites RhoA/Rac1/Cdc42 to analyze their role in expression and regulation of cyclooxygenase-2 (COX-2) in endothelial cells (EC). Pretreatment of EC with TcdB-10643 prevented lipopolysaccharide (LPS)- or tumor necrosis factor- (TNF )-related COX-2 expression but had no effect on COX-1 protein levels. TcdB-10463 preincubation suppressed LPS-dependent nuclear factor-B activation (NF- B). Rho inhibition did not affect COX-1 activity. Inactivation of Rho proteins before LPS stimulation blocked arachidonic acid (AA)-, thrombin-, and Escherichia coli hemolysin (HlyA)-dependent release of COX-2-related 6-ketoprostaglandin F₁ (6k-PGF₁). In contrast, Rho inhibition did not affect COX-2-dependent 6k-PGF₁ liberation when TcdB-10643 was added 10 h after LPS or TNF stimulation of EC. Therefore, RhoA/Rac1/Cdc42 contribute to NF- B-dependent LPS- and TNF-induced expression of PGHS-2 in EC but had no effect on the activity of expressed COX-1 and COX-2.     

Src-Family Kinase-p53/56lyn Plays an Important Role in TNFα-Stimulated Production of O2 − by Human Neutrophils Adherent to Fibrinogen

Stimulation of neutrophil function by TNF is largely dependent on ₂ integrins. It has also been proposed that src-family kinases are involved in this process. However, the functions of src-like kinases in human neutrophils still remain to be determined. In the present study, we used the new src-family kinase specific inhibitor PP1 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine to investigate the role src-kinases play in TNF stimulation of neutrophil function. Our results demonstrated that, in neutrophils adherent to fibrinogen, PP1 inhibited TNF-stimulated superoxide production and protein tyrosine phosphorylation in a dose-dependent manner. In in vitro kinase assays, PP1 profoundly inhibited the activation of p53/56^lyn but not p59^hck or p72^syk. Only slight inhibition was found of p58^c-fgr. These data indicate that p53/56^lyn plays an important role in TNF-mediated stimulation of PMN function.     

Effects of C5a and FMLP on Interleukin-8 Production and Proliferation of Human Umbilical Vein Endothelial Cells

Interleukin-8 (IL-8), C5a and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are chemotactic peptides with predominant effects on leukocytes during inflammation. With emphasis on C5a we studied the regulation of the production of IL-8 by human umbilical vein endothelial cells (HUVEC) in vitro. Primary HUVEC cultures were incubated with IL-1, TNF, C5a and fMLP for 24 h and 48 h prior to measurement of IL-8 in supernatants of the cells by an enzyme immunoassay. Whereas IL-1 and TNF significantly increased the levels of IL-8, C5a decreased the IL-8 production after 48 h. In addition, the ability of IL-1, TNF, C5a, fMLP and IL-8 to induce cell proliferation was compared by means of a ³H-thymidine incorporation assay. In contrast with IL-1 and TNF, both C5a and fMLP increased cell proliferation of HUVEC. This increase occurred with increasing concentrations of C5a contrary to IL-8, which showed increased cell proliferation at low, but not high IL-8 concentrations.     

In Vitro and In Vivo Effects of Genistein on Murine Alveolar Macrophage TNFα Production

The present study was performed to determine whether genistein could inhibit in vivo LPS-induced alveolar macrophage TNF production and thus reduce the alveolar neutrophil influx following LPS. In vitro incubation with genistein completely inhibited LPS-induced TNF production by alveolar macrophages (AM) from BALB/c mice. Subsequently mice were pretreated with intraperitoneal genistein or vehicle, then received nasal LPS to induce an alveolitis. Genistein was then administered every eight hours for five days following LPS. At 24 hours after LPS, the bronchoalveolar lavage (BAL) TNF and ex vivo TNF production from AM, were lower in the genistein treated animals. As well, total BAL white blood cell (WBC) count was reduced in the genistein as compared to the vehicle-only group. The percent neutrophils and the resolution of neutrophils were similar between genistein and vehicle groups. Therefore, genistein was able to decrease AM TNF production, and was associated with a decrease in BAL WBC count post-LPS.     

Tumor necrosis factor alpha production in schistosomiasis with carcinoma of urinary bladder

Schistosomiasis parasitic infection (Schistosoma haematobium) is associated in some patients with bladder cancer. The production of cytokines such as tumor necrosis factor alpha (TNF) is a key event of inflammation in human infectious disease and malignancy. TNF has not been previously investigated from schistosomiasis infection and bladder malignancy. In this report we demonstrate that serum levels of TNF are highly elevated in patients with schistosomiasis of urinary bladder (SB), schistosomiasis with carcinoma of urinary bladder (SCB), and carcinoma of urinary bladder without schistosomiasis (CB). Purified monocytes from bladder malignancy (SCB and CB) cultured without exogeneous stimuli release TNF in the culture supernatants. However, lipopolysaccharides and concanavalin A stimulation of monocytes from these patients produced highly elevated levels of TNF compared with normal controls. The findings that monocytes are the potent producers of TNF in this malignancy may be a key observation implicating these cells in the pathophysiology of this disease. Furthermore, it was shown that serum TNF levels correlated with the clinical staging of disease in both SCB and CB, with higher levels in T3 and T4 advanced-stage patients and low levels in T1 and T2 early-stage patients. These results suggest that monocyte abnormality and serum TNF levels might be one of the factors contributing to the progression of disease.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

A novelin vitro assay system for transendothelial tumor cell invasion: significance of E-selectin and α3 integrin in the transendothelial invasion by HT1080 fibrosarcoma cells

The interaction of tumor cells with endothelial cells is a key event in tumor metastasis. We established anin vitro invasion assay system, in which the invasion of tumor cells after interaction with endothelial cells can be examined. Two chamber culture wells separated by porous membrane were used. Human umbilical vein endothelial cells (HUVEC) were placed on porous membranes coated with matrix components. The invasion by HT1080 fibrosarcoma cells was determined in this system by counting the number of cells that moved through the membranes from upper to lower chambers. HUVEC cells did not migrate through the membranes as judged by the staining with UEA-I. Observation by scanning electron microscopy revealed that HT1080 cells bound to HUVEC surfaces and migrated underneath the HUVEC monolayer. Effects of antibodies specific for cell surface adhesion molecules on the migration of HT1080 cells were examined. Invasion of uncoated membranes and membranes coated with HUVEC cells was compared. Antibody against E-selectin significantly suppressed an increase of HT1080 cell invasion of HUVEC monolayers stimulated by IL-1 or TNF. Antibody against integrin ₃ subunit remarkably inhibited the invasion of HUVEC cell-coated membranes, suggesting that integrins with the ₃ subunit may play an important role in the transendothelial invasion by HT1080 cells.     

Rat Macrophage Inflammatory Protein-1α, a CC Chemokine, Acts as a Neutrophil Chemoattractant in Vitro and in Vivo

Recombinant rat macrophage inflammatory protein-1 (rMIP-1) at a concentration of 3 × 10^–8 M had strong neutrophil chemotactic activity, though the potency of rMIP-1 was less than that of cytokine-induced neutrophil chemoattractant (CINC)-1 at lower concentrations. In addition, rMIP-1 induced neutrophil chemotaxis in vivo when rMIP-1 was injected into the preformed air-pouch on the back of rats. The adhesion of rMIP-1-treated neutrophils to fibrinogen significantly increased, reaching a maximum adhesion at 10^–8 M. Stimulation of neutrophils with rMIP-1 induced a transient increase in intracellular free [Ca²⁺] dose-dependently. rMIP-1 still induced an increase in the intracellular [Ca²⁺] of rat neutrophils stimulated first with CINC-1, CINC-3 or C5a, suggesting that rat neutrophils have a specific receptor for rMIP-1. Supporting these findings, an additive increase in chemotactic potency was found when both rMIP-1 and CINC-1 were added to the lower wells of Boyden chamber in vitro. In addition, high levels of rMIP-1 were detected in the inflammatory site of air-pouch/carrageenan-induced inflammation in rats. Our results suggest that rMIP-1 acts as a neutrophil chemoattractant and, together with CINCs, plays an important role in infiltration of neutrophils into inflammatory sites in rats.     

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1) and tumor necrosis factor alpha (TNF) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1 and TNF were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 and TNF significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 and TNF displayed significant in vivo antitumor activity against the IL1- and TNF-resistant B16F10 metastatic murine melanoma.     

Potential roles for tumour necrosis factorα during embryonic development

This paper reviews the evidence indicating possible roles for tumour necrosis factor-alpha (TNF) in development. It is proposed that TNF may have essentially three major roles during embryonic development, which may be analogous to its roles in the immune system and during inflammation: a role in programmed cell death; a role as a cellular growth and differentiation factor; and also a role in the remodelling of extracellular matrix, and the regulation of cell adhesion molecules and integrins. The concept of the existence of a cytokine array during embryogenesis, analogous to that occurring in inflammation, is discussed, as well as potential roles for TNF in the induction of ubiquitin; protective mechanisms embryonic cells may employ against TNF-mediated cytotoxicity; and a consideration of the role TNF may play in a free radical theory of development.     

Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration “in vivo” by indirect mechanisms

The and forms of recombinant interleukin-1 (IL-1 and IL-1) and of recombinant Tumor Necrosis Factor (TNF and TNF) induced dose-dependent neutrophil migration into rat peritoneal cavities. Migration induced by both IL-1s showed a bell-shaped dose-response curve and IL-1 was 3-fold more potent than IL-1. Pretreatment of the animals with dexamethasone or depletion of the peritoneal macrophage population, abolished the neutrophil migration induced by the four cytokines. In vitro stimulation of macrophage monolayers with IL-1 and the TNFs released a factor into the supernatant which, unlike these cytokines, induced neutrophil migration in dexamethasone pretreated animals. These results suggest that the neutrophil migration induced by IL-1, IL-1 and TNF is not due to a direct effect on neutrophils, but occurs via the release of a chemotactic factors(s) from resident macrophages.     

Defective expression of FasL and Bax in human lung cancer

Abstract. The present studies investigated whether FasL and Bax genes are expressed in pleuro-pulmonary biopsies from patients with lung cancer. FasL, Bax, and TNF mRNAs were detected in 19 biopsies of primary or metastasic lung cancer by fluorescent in situ hybridization assays. Fluorescent probes were produced by polymerase chain reaction using a human spleen lambda gt11 library and specific primers for FasL, Bax, and TNF. Proteins were detected by immunohistochemistry using monoclonal anti- FasL, anti-Bax, and anti-TNF antibodies. Chromatin fragmentation was detected by TUNEL. Seven negative samples from subjects without lung pathology were obtained during legal autopsies and 12 positive control biopsies from patients with lung infections were also included. Sixty-eight percent of lung cancer biopsies exhibited FasL; Bax was expressed in 68% and TNF in 63%. FasL protein was detected in 21%, Bax protein in 26%, and TNF was present in 31% of cancer biopsies. A low degree of apoptosis in lung cancer was demonstrated by TUNEL assays. A defect in FasL, Bax, and TNF gene expression was found in lung cancer biopsies. Some tumors normally expressed the mRNA of FasL, Bax, or TNF, but their proteins were absent, or were non-functional, since TUNEL assays were negative. Such a failure would contribute to cancer cell survival and dissemination.     

Immunohistochemical localization of αl-antitrypsin and αl-antichymotrypsin in salivary pleomorphic adenomas

Summary Immunohistochemical identification of l-antitrypsin (l-AT) and l-antichymotrypsin (l-ACh) in pleomorphic adenomas of salivary glands is reported in order to compare their distribution profiles with those of lysozyme and lactoferrin, already described elsewhere.Normal salivary glands indicated positive l-AT staining in ductal segments and had no l-ACh in any glandular cell. Pleomorphic adenomas displayed moderate positivity to l-AT staining in duct-like, tubular and glandular epithelia which was particularly intense in luminal cells. The limited number of tumour cells which showed duct-like structures with a single cellular layer arrangement, displayed the highest staining to l-ACh. Strongly l-AT positive tumour cells located on the inner side of luminal cavities were also markedly positive to l-ACh. Spindle shaped tumour cells existed outside tubular and ductal structures and were negative to l-AT and l-ACh.Distribution of l-AT in salivary glands was similar to that of lysozyme as is usual in ductal segments or their transformed cells, and occurrence of l-ACh localization rather resembled that of lactoferrin, with occurrence in acinar compartments and changed epithelia within acini.The biological role of a specific immunohistochemical distribution of l-AT and l-ACh in pleomorphic adenomas may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.     

Expression of matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) induced by tumour necrosis factor α correlates with metastatic ability in a human osteosarcoma cell line

We have examined the correlation between matrix metalloproteinase (MMP) expression and metastatic properties of a low metastatic osteosarcoma cell line, osteosarcoma takase (OST), under stimulation by tumour necrosis factor (TNF). In vivo, OST cells exhibited significantly increased colonization in the lungs of nude mice in a dose-dependent manner when they were treated by TNF prior to injection. In vitro, TNF enhanced tumour cell invasion through the reconstituted basement membrane in a transwell chamber up to 2.5-fold. Gelatin zymography and sandwich enzyme immunoassays demonstrated marked production of MMP-9 [92-kDa gelatinase/type IV collagenase (gelatinase B)] but not MMP-2 [72-kDa gelatinase/type IV collagenase (gelatinase A)], MMP-3 (stromelysin-1) or MMP-7 (matrilysin). Motility of the tumour cells and adhesion to cultured endothelial cells were slightly increased by the TNF treatment up to 1.6-fold and 1.4-fold, respectively, while the growth rate was decreased. These results suggest that upregulation of MMP-9 together with enhanced motility and endothelial adhesion contribute to the increased metastatic ability of OST cells induced by TNF treatment.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Triggering of respiratory burst by tumor necrosis factor in neutrophils adherent to fibronectin. Evidence for a crucial role of CD18 glycoproteins

Human neutrophils, plated on fibronectin (FN)-coated wells, were found to release large quantities of superoxide anion (O ₂ ^– ) in response to tumor necrosis factor alpha (TNF-). The O ₂ ^– release was completely inhibited by two monoclonal antibodies (MoAbs, MHM23 and TS1/18) against CD18 glycoproteins. An independently derived anti-CD18 MoAb (60.3) was ineffective. These MoAbs failed to inhibit neutrophil adhesion to FN-coated surfaces. Moreover, neutrophils incubated for 30 min on FN and then washed to remove non-adherent cells, were responsive to TNF- in the presence of anti-CD18 MoAbs MHM23 and TS1/18. Consequently, the CD18-dependent capacitation of the respiratory burst can occur before TNF- triggering. Finally, neutrophils plated on FN in the presence of anti-CD18 MoAbs and then washed, i.e. adherent cells blocked in their surface CD18 molecules, released O ₂ ^– after adding TNF- but only in the absence of additional anti-CD18 MoAbs. This is consistent with a TNF- ability to induce rapid expression and activation of new oxidative burst-capacitating CD18 molecules. The results suggest that the anchorage of neutrophils to FN surfaces depends on adherence molecules apparently unrelated to CD18, probably the so-called fibronectin receptors (FNRs), whereas the capacitation of the respiratory burst in response to TNF- requires the intervention of CD18 glycoproteins, available on the membrane of resting neutrophils or mobilized to the cell surface by TNF-.     

Immunoreactivity of the JK-132 monoclonal antibody directed against basement membrane collagen in normal and diabetic glomeruli

The possible involvement of basement membrane-associated collagen (recognized by the monoclonal antibody JK-132) in the evolution of diabetic nephropathy was studied in kidney specimens from seven patients with noninsulin-dependent diabetes mellitus, and its distribution was compared with those of antibodies against 1 to 4 chains of type IV collagen. JK-132, a monoclonal antibody against basement membrane-associated collagen, reacted immunohistochemically exclusively with the mesangial matrix of the glomerular capillary. In contrast, antibodies to the 1 and 2 chains (IV) reacted strongly with mesangial matrix, and less strongly with the glomerular basement membrane (GBM). Antibodies to the 3 and 4 chains (IV) reacted mainly with GBM. In diabetes, JK-132 reacted most extensively with the expanded mesangial matrix, its staining intensity increasing with progression of the diabetic glomerulosclerosis. Antibodies to the 1 and 2 chains (IV) reacted prominently with the expanded mesangial matrix but less strongly with the GBM. Antibodies to the 3 and 4 chains reacted intensely with the thickened GBM. These results suggest that basement membrane-associated collagen differs from 1 to 4 chains of type IV collagen and that basement membrane-associated collagen is a good marker of mesangial expansion in diabetic nephropathy.