Modulation of porcine intestinal epitheliocytes immunetranscriptome response by Lactobacillus jensenii TL2937.

In order to evaluate probiotic strains applicable for the beneficial immunomodulation of the porcine gut (immunobiotics), we previously developed a porcine intestinal epitheliocyte cell line (PIE cells). Here, transcriptomic studies using PIE cells were performed considering that this information would be valuable for understanding the mechanisms involved in the protective activity of the immunobiotic strain Lactobacillus jensenii TL2937 against intestinal inflammatory damage in pigs. In addition, those studies would provide criteria for selecting biomarkers for the screening of new immunobiotic strains. We performed microarray analysis to investigate the transcriptomic response of PIE cells to the challenge with heat-stable enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs) and, the changes induced by L. jensenii TL2937 in that response. The approach allowed us to obtain a global overview of the immune genes involved in the response of PIE cells to heat-stable ETEC PAMPs. We observed that L. jensenii TL2937 differently modulated gene expression in ETEC PAMPs-challenged PIE cells. Microarray and RT-PCR analysis indicated that the most remarkable changes in PIE cells transcriptomic profile after heat-stable ETEC PAMPs challenge were observed in chemokines, adhesion molecules, complement and coagulation cascades factors. In addition, an anti-inflammatory effect triggered by TL2937 strain in PIE cells was clearly demonstrated. The decrease in the expression of chemokines (CCL8, CXCL5, CXCL9, CXCL10, and CXCL11), complement (C1R, C1S, C3, and CFB), and coagulation factors (F3) by L. jensenii TL2937 supports our previous reports on the immunoregulatory effect of this strain. These results provided clues for the better understanding of the mechanism underlying host-immunobiotic interaction in the porcine host. The comprehensive transcriptomic profiles of PIE cells provided by our analyses successfully identified a group of genes, which could be used as prospective biomarkers for the screening and evaluation of new anti-inflammatory immunobiotics for the prevention of inflammatory intestinal disorders in pigs.

Erythromycin attenuates an experimental model of chronic bronchiolitis via augmenting monocyte chemoattractant protein-1.

The mechanisms underlying the therapeutic efficacy of erythromycin (EM) in diffuse panbronchiolitis (DPB) was investigated. For this purpose, an experimental rabbit model of DPB induced by Pseudomonas aeruginosa inoculation was employed. Daily administration of EM (3 mg x kg x day(-1)) led to an increase in the number of macrophages in bronchoalveolar lavage fluid (BALF) at an early phase, while reducing the size of granulomatous lesions at the late phase without affecting the number of viable bacteria recovered from the infected lung. Reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunohistochemical studies showed that monocyte chemoattractant protein (MCP)-1 was produced in both BALF and infected lung. EM treatment resulted in a significant increase in the level of MCP-1 in BALF, while reducing that of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-8. EM also increased MCP-1 messenger ribonucleic acid (mRNA) and protein expression in the infected lung. MCP-1 blockade abolished the protective effect of EM, as neutralization of MCP-1 with anti-MCP-1 antibodies reduced the EM-induced increase in the number of macrophages in BALF, and augmented size of the granulomatous lesions, as compared to control. The results of the present study suggest that erythromycin attenuates the pulmonary granuloma formation, at least in part, by increasing the production of monocyte chemoattractant protein-1.

Cardiac amyloidosis presenting as microvascular angina--a case report.

Cardiac amyloidosis usually presents with heart failure, but rarely leads to coronary insufficiency. The authors report a case of a 69-year-old Japanese woman with cardiac amyloidosis presenting as microvascular angina. She had exertional angina with positive exercise test and normal coronary angiograms. However, heart failure developed, and she died 3 years after symptom onset. On autopsy, coronary arteries were patent. In contrast to that of the epicardial coronary arteries, histologic examination of the heart revealed severe obstructive alterations of the intramural coronary arteries with amyloid. Furthermore, amyloid was present mainly in the endocardium and the intramural coronary arteries, but there was little present in the myocardium. This case was a rare AL amyloidosis. There have been only 4 reported cases of cardiac amyloidosis that presented with exertional angina, a positive exercise test, and normal coronary angiograms and histologic examination.

Regulation of H(+)-ATPase synthesis in response to reduced pH in ruminal bacteria.

The capacity of ruminal bacteria to regulate H(+)-ATPase synthesis in response to reduced pH was investigated to explain acid tolerance. The activity of H(+)-ATPase in Streptococcus bovis, an acid-tolerant bacterium, was 2.2-fold higher at pH 4.5 than at pH 5.5. The increase in the amount of H(+)-ATPase protein was similar, suggesting that the increase in H(+)-ATPase activity is owing to the increase in H(+)-ATPase synthesis. The level of atp-mRNA at pH 4.5 was 2.5-fold higher than at pH 5.5, indicating that H(+)-ATPase synthesis is regulated at the transcriptional level, responding to low pH. In Ruminococcus albus, an acid-sensitive bacterium, H(+)-ATPase activity, the amount of H(+)-ATPase protein, and the level of atp-mRNA at pH 7.0 were similar to the values at pH 6.0, the lowest pH permitting growth. This result suggests that R. albus is incapable of enhancing H(+)-ATPase synthesis at low pH. Thus, acid tolerance appeared to be related to the capacity to augment the synthesis of H(+)-ATPase responding to low pH.

Cloning and characterization of 5'-flanking region of mouse prostacyclin synthase gene.

To gain an insight into the mechanisms of prostacyclin expression, a genomic DNA clone harboring 2.0 kb of the 5'-flanking sequence of the mouse prostacyclin synthase (PGIS) gene was isolated. The 5'-flanking region did not possess a TATA box, but contained a GC-rich region and several consensus cis DNA elements. The major product of the primer extension analysis suggested that the transcription of the gene started from 72 bases upstream of the translational initiation codon. To analyze the PGIS promoter activity, the 2.0 kb fragment was fused to the luciferase gene and transient transfection assays were conducted with cultured rat vascular smooth muscle cells (VSMC). The fragment showed significant promoter activity in the cells. Analysis of a series of 5'-deletion constructs showed that the 5'-flanking regions spanning bases -371 to -285 and -229 to -119 were important for the basal transcriptional activity of the mouse PGIS gene. Gel mobility shift assays revealed that DNA-protein complexes were formed with the nuclear extracts from VSMC, and that the formation of these complexes was inhibited by excess consensus Sp1 oligonucleotide. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in supershift of the band for the DNA-protein complex. In addition, mutation of two Sp1 recognition motifs residing at bases -297 to -289 and -197 to -192 markedly reduced the basal PGIS promoter activity and retarded the band in a gel mobility shift assay. These results indicated that binding of one Sp1 to two Sp1 sites on the promoter region activated the basal transcription of the PGIS gene.

CXC chemokine GRO is essential for neutrophil infiltration in LPS-induced uveitis in rabbits.

The purpose of this study was to investigate the role and regulation of the CXC chemokine GRO and the interaction between GRO and IL-8 in LPS-induced uveitis in rabbits. Uveitis was induced by intravitreal injection of 100 ng of LPS in rabbits. After the LPS injection, GRO was produced in aqueous humor and peaked at 24 hr. Immunohistochemistry showed that ciliary epithelial cells were responsible for production of GRO. Blocking the activity of GRO by anti-GRO serum reduced LPS-induced aqueous neutrophil counts by 80%, but did not reduce the mononuclear cell counts or protein levels or IL-8 levels. Regulation of GRO production by TNFalpha, IL-1 and IL-8 was studied. Anti-TNFalphamAb alone did not inhibit the 24 hr LPS induced GRO levels, whereas rrIL-1Ra inhibited the GRO production by 58%. The combination of anti-TNFalpha mAb and rrIL-1Ra inhibited 93% of GRO production. Although treatment with anti-IL-8 IgG inhibited the neutrophil infiltration by 66%, treatment with this antibody did not inhibit GRO production. Taken together, our results suggest that GRO is an essential mediator for neutrophil infiltration in LPS-induced uveitis in rabbits. Most of GRO production is mediated by TNFalpha and IL-1. GRO and IL-8 act in concert to mediate neutrophil infiltration.

Neutrophil infiltration as a crucial step for monocyte chemoattractant protein (MCP)-1 to attract monocytes in lipopolysaccharide-induced arthritis in rabbits.

OBJECTIVE AND DESIGN: To evaluate the mechanism whereby monocyte chemoattractant protein (MCP)-1 attracts monocytes in vivo. SUBJECTS: New Zealand white rabbits (175 rabbits) were used. TREATMENT: LPS, MCP-1 or IL-8 was injected into knee joints. Antibodies against various cytokines or IL-1 receptor antagonist were injected to neutralize cytokine activities. METHODS: The numbers of leukocyte populations, levels of cytokines in joints were estimated. RESULTS: Partial inhibition of neutrophil influx with anti-IL-8 IgG (10 microg) suppressed LPS-induced macrophage influx by 43 +/- 8.5% (p<0.05) without affecting the MCP-1 level. Intraarticular injection of MCP-1 (1-30 microg) induced macrophage influx. The event was accompanied by a small number of neutrophils in an early phase. Co-injection of IL-8 (1.0 microg) enhanced the MCP-1-induced macrophage infiltration (p < 0.01). In neutrophil-depleted rabbits, LPS failed to induce macrophage influx even though the MCP-1 level was maintained, and macrophage influx following exogenously administered MCP-1 was also dramatically inhibited. CONCLUSIONS: Early events associated with neutrophil infiltration appear to be important for MCP-1 to induce a later macrophage influx in LPS-arthritis.

Comparison of endometrial carcinoma coexisting with and without endometrial hyperplasia.

OBJECTIVE: It has been reported that endometrial carcinoma has two pathogenic types; one with and the other without endometrial hyperplasia. The present study was done to clarify whether this was true in our series of a large number of Japanese patients. METHODS: Three hundred and twenty-eight patients with endometrial carcinoma were classified into two groups, one coexisting endometrial hyperplasia (group 1; 182 cases) and the other having normal endometrium without endometrial hyperplasia (group 2; 107 cases). The clinical and pathological observations of the two groups were compared. RESULTS: The ages of the patients were significantly lower in group 1 (30-83; mean 50.7) than in group 2 (34-81; mean, 57.2). The incidence of the carcinoma with invasion limited to less than 1/3 of the myometrium, with surgical stage I, lower grade endometrioid type and higher levels of progesterone receptor, and without lymph node metastasis were significantly higher in group 1 than in group 2. The cumulative survival rate for stage I was also significantly higher in group 1 patients. CONCLUSION: A significant difference was found between groups 1 and 2 in age distribution, degree of progress and pathological observations. Stage I patients in the former group had a better prognostic than the latter.

Involvement of growth-related protein in lipopolysaccharide-induced rabbit arthritis: cooperation between growth-related protein and IL-8, and interrelated regulation among TNFalpha, IL-1, IL-1 receptor antagonist, IL-8, and growth-related protein.

We investigated the functional role of a CXC chemokine, growth-related protein (GRO), in the recruitment of neutrophils in lipopolysaccharide (LPS)-induced rabbit arthritis. The amounts of GRO in the synovial fluids (SF) reached the first peak (major) at 2 hours and the second peak (minor) at 9 hours after injection of LPS into the knee joints. Administration of anti-GRO mouse monoclonal antibody inhibited 54% of the peak leukocyte accumulation at 9 hours (neutrophils greater than 95%), which was similar to the inhibition by anti-IL-8 IgG (48%). Co-administration of these inhibitors increased the inhibition up to 70% at 9 hours and also inhibited 65% of the initial phase of leukocyte infiltration at 2 hours (neutrophils greater than 99%), which was not affected by a single administration of each inhibitor. The amounts of GRO in SF at 2 hours were not altered by either anti-TNFalpha mAb or anti-IL-8 IgG, but reduced by rabbit recombinant IL-1 receptor antagonist (rrlL-1Ra) by 39%. The inhibition by rrlL-1 Ra was augmented further to 59% with coadministered anti-TNFalpha mAb. In contrast, the amounts of GRO at 9 hours were reduced by rrlL-1Ra by 67%. There was no additional reduction in the amounts of GRO at 9 hours by either combination of rrlL-1Ra with anti-TNFalpha mAb or anti-IL-8 IgG. Administration of anti-GRO mAb did not alter TNFalpha or IL-8 contents in SF at their peak (2 hours), but reduced the amounts of IL-1beta at 6 hours and IL-1Ra at 9 hours by 42% and 49%, respectively. These results provide evidence for the following: (a) GRO as well as IL-8 are important mediators involved in the recruitment of neutrophils both in the early and the late phase of LPS-induced arthritis, (b) IL-1 produced in the early phase stimulates GRO production, (c) GRO plays a role in the later induction of IL-1beta and IL-1Ra, and (d) induction of GRO is not regulated by IL-8.

Role and regulation of IL-8 and MCP-1 in LPS-induced uveitis in rabbits.

Intravitreal injection of lipopolysaccharide (LPS) induces leukocyte infiltration and protein leakage into the aqueous humor. In the present study, we investigated the role of IL-8 and MCP-1 and regulation of these chemokines by TNFalpha and IL-1 in LPS-induced uveitis in rabbits. After intravitreal injection of LPS, generation of IL-8 in the aqueous humor showed a biphasic pattern with the first peak at 12 hr and the second one at 24 hr, while MCP-1 was produced in a monophasic pattern and peaked at 24 hr. Immunohistochemistry showed that ciliary epithelial cells and infiltrating leukocytes were the producing cells of IL-8 and MCP-1. Administration of anti-IL-8 IgG suppressed by 66% the peak levels of LPS-induced aqueous neutrophil counts at 24 hr but did not suppress aqueous mononuclear cell counts or protein levels. anti-MCP-1 IgG inhibited aqueous mononuclear cell counts by 41% and protein levels by 28%, but did not inhibit aqueous neutrophil counts. The levels of LPS-induced aqueous IL-8 and MCP-1 at 12 hr were inhibited by anti-TNFalpha mAb but not by an IL-1 receptor antagonist (IL-1Ra), while concentrations of the two chemokines at 24 hr were inhibited by both anti-TNFalpha mAb and IL-1Ra. A combination of anti-TNFalpha mAb and rrIL-1Ra had an additive effect on the 24 hr-chemokine levels and inhibited up to 90% chemokine production. Taken together, our results show that IL-8 mediates neutrophil infiltration, while MCP-1 mediates mononuclear cell infiltration and protein leakage in LPS-induced uveitis in rabbits. Levels of aqueous IL-8 and MCP-1 at 12 hr are regulated by TNFalpha, while levels at 24 hr are regulated by TNFalpha and IL-1.

Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8.

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.

Involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist in LPS-induced rabbit uveitis.

The objective of the study was to investigate involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist (IL-1Ra) in lipopolysaccharide (LPS)-induced uveitis. Intravitreal injection of LPS (100 ng) to rabbits induced a massive leukocyte infiltration and protein leakage into the aqueous humor. Aqueous leukocyte counts and protein levels reached a peak 24 hr after this injection. The peak concentrations of aqueous TNF alpha (230 +/- 37 pg ml-1, at 9 hr) and IL-1 beta (185 +/- 80 pg ml-1, at 18 hr) preceded peak levels of aqueous leukocyte counts and protein levels. In contrast, the levels of aqueous IL-1Ra peaked at 48 hr (12,239 +/- 1964 pg ml-1) and a fairly high concentration of IL-1Ra remained when the inflammatory reactions subsided. Immunohistochemistry and leukocyte-depletion studies showed that infiltrating leukocytes were the major cellular sources of aqueous TNF alpha, IL-1 beta and IL-1Ra. Intravitreal injection of homologous TNF alpha (0.1-1.5 micrograms) or IL-1 beta (0.5-5 ng) reproduced a rapid leukocyte infiltration and protein leakage. Administration of anti-TNF alpha mAb (10 micrograms) suppressed the number of LPS-induced infiltrating neutrophils by 50%, mononuclear cells by 58%, and protein leakage by 42%. Administration of rabbit IL-1Ra (10 micrograms) also suppressed neutrophil influx by 78%, however, neither mononuclear cell influx nor protein leakage was inhibited by rabbit IL-1Ra. Co-administration of the two inhibitors enhanced inhibition of neutrophil infiltration to 88%, and protein leakage to 64%. We conclude that TNF alpha and IL-1 beta are the principal mediators of LPS-induced uveitis. Our observations also suggest that endogenous IL-1Ra may down-regulate inflammatory reactions.

Analysis of the cytokine network among tumor necrosis factor alpha, interleukin-1beta, interleukin-8, and interleukin-1 receptor antagonist in monosodium urate crystal-induced rabbit arthritis.

In the present study, we analyzed the cytokine network among TNFalpha, IL-1beta, IL-8, and IL-1 receptor antagonist (IL-1Ra) in a rabbit experimental model of acute gout. The production of TNFalpha in synovial fluids reached the peak at 2 hours after the intra-articular injection of monosodium urate (MSU) crystals. The production of IL-1beta and IL-8 reached the first peak at 2 hours and the second peak at 9 and 12 hours, respectively. The production of endogenous IL-1Ra reached the peak at 9 hours. The source of TNFalpha and the first phase of IL-8 was synovial cells, whereas infiltrating leukocytes were the source of the second phase of IL-8 and also of IL-1beta and IL-1Ra. The production of TNFalpha was not altered by either anti-lL-8 IgG or IL-1Ra. The first IL-1beta peak was reduced only with a combination of anti-TNFalpha mAb and anti-lL-8 IgG, whereas the second peak was significantly reduced by either inhibitor. The first IL-8 peak was not altered with anti-TNFalpha mAb or IL-1 Ra, whereas the second IL-8 peak was reduced with IL-1Ra. Anti-TNFalpha mAb or anti-lL-8 IgG significantly reduced the peak level of endogenous IL-1Ra. These cytokine inhibitors also attenuated the maximal leukocyte accumulation at 9 hours, but not the initial phase, which occurred within 2 hours. These results provide evidence that IL-8 and TNFalpha were responsible for the production of IL-1beta and IL-1Ra, and that IL-1beta was responsible for the second phase of IL-1beta and IL-8 production. Our data also suggest that the initial and the maximal phases of leukocyte influx are differently regulated. Finally, the intravenous injection of colchicine inhibited neutrophil infiltration without affecting the production of TNFalpha or the first peak of IL-8, suggesting that colchicine inhibits MSU crystal-induced arthritis by directly inhibiting the migration of neutrophils.

IL-8 is an essential mediator of the increased delayed-phase vascular permeability in LPS-induced rabbit pleurisy.

We investigated the involvement of IL-8 in the delayed vascular permeability (VP) in rabbit lipopolysaccharide (LPS)-pleurisy. Maximal level of interleukin-8 (IL-8) was detected in pleural fluid at 2 h after LPS injection and anti-IL-8 inhibited the delayed VP by 90%. Injection of homologous IL-8 induced VP, the time-course of which preceded that of LPS-induced delayed VP. Production of IL-8 in LPS-pleurisy was inhibited with anti-tumor necrosis factor alpha (TNF-alpha), whereas the production of TNF-alpha was not affected with anti-IL-8. Injection of IL-8 did not induce TNF-alpha production and anti-TNF-alpha had no effect on IL-8-induced VP. Injection of homologous TNF-alpha induced IL-8 production and VP, and TNF-alpha-induced delayed VP was blocked with anti-IL-8. These results indicate important roles of IL-8 in LPS-induced delayed VP and that TNF-alpha causes the delayed VP through the production of IL-8.

Analysis of the inflammatory cytokine network among TNF alpha, IL-1 beta, IL-1 receptor antagonist, and IL-8 in LPS-induced rabbit arthritis.

We investigated the cytokine network in rabbit lipopolysaccharide (LPS)-induced arthritis, using inhibitors against homologous TNF alpha, IL-1 beta, and IL-8. Rabbits were intraarticularly injected with LPS (10 ng) and cytokine inhibitors (10 micrograms each), and the concentrations of each cytokine in the synovial fluids were measured. Maximum levels of TNF alpha and IL-8 were detected at 2 hours after LPS-injection, whereas IL-1 beta and IL-1 receptor antagonist (IL-1Ra) were detected at 6 and 9 hours, respectively. By immunohistochemistry, synovial lining cells were positive for TNF alpha and IL-8, and infiltrating leukocytes were positive for IL-1 beta and IL-1 Ra. The effects of cytokine inhibitors on the release of each cytokine were then investigated. The maximum levels of TNF alpha and IL-8 were not affected by blocking the activities of other cytokines. In contrast, the peak concentration of IL-1 beta was reduced by anti-TNF alpha monoclonal Ab (mAb), IL-1 Ra or anti-IL-8 IgG. Peak concentrations of IL-1Ra were reduced by anti-TNF alpha mAb or anti-IL-8 IgG. Anti-TNF alpha mAb, IL-1Ra, and anti-IL-8 IgG reduced the recruitment of leukocytes into the joint cavity, and the effect of anti-IL-8 IgG was less than that of anti-TNF alpha mAb plus IL-1Ra. The initial phase of the leukocyte influx was not inhibited. These results provide new evidence that IL-8 as well as TNF alpha are the most proximal cytokines and induce subsequent production of IL-1 beta and IL-1Ra. The data also raise the possibility that factor(s) other than IL-8 may be involved in the leukocyte influx in LPS-induced arthritis.

Detection and characterization of IL-1 receptor antagonist in tissues from healthy rabbits: IL-1 receptor antagonist is probably involved in health.

Interleukin 1 (IL-1) is postulated to function in maintaining homeostasis, however, over-action of this cytokine may lead to disruption of homeostasis due to it's wide spectrum of activities. To understand the endogenous regulation of this cytokine, we examined the existence of IL-1 receptor antagonist (IL-1Ra) in tissues from healthy rabbits. IL-1Ra was constitutively produced in all tissues examined (lung, liver, spleen, thymus, caecum, skin, kidney, heart, and brain), as estimated by ELISA. Immunoprecipitation, RT-PCR and immunohistochemical studies indicated that all tissues produced secreted form of IL-1Ra (sIL-1Ra), whereas thymus, caecum, skin and kidney produced both sIL-1Ra and intracellular of IL-1Ra. All tissue IL-1Ra purified using anti-IL-1Ra IgG affinity chromatography had inhibitory activity on the IL-1-induced thymocyte proliferative response, and the activity was totally abolished by anti-IL-1Ra mAb. No IL-1 activity was detected in any tissues except skin and heart, however, after preincubation of the samples with anti-IL-1Ra, the activity was first visible in the tissues. Under these conditions, IL-1 activity in skin and heart was enhanced to 170% and 280%, respectively. Taken together, we conclude that tissue IL-1Ra is involved in health maintenance by masking co-existing IL-1 activity present in tissues.

Lead-induced alteration of heparan sulfate proteoglycans in cultured vascular endothelial cells.

We investigated the alterations of heparan sulfate proteoglycans produced by vascular endothelial cells after exposure to lead. Bovine aortic endothelial cells were cultured and metabolically labeled with [3H]glucosamine and [35S]sulfate in the presence of lead chloride at 10 microM. Radiolabeled HSPGs were separated by ion-exchange chromatography and either their hydrodynamic size or the length of heparan sulfate chains were characterized by gel filtration. It was found that lead markedly suppresses the incorporation of the radioactive precursors into HSPGs in the cell layer; the incorporation into chondroitin/dermatan sulfate proteoglycans was decreased by the metal only slightly. The suppression by lead occurred in the low molecular weight subclass of HSPGs rather than the high molecular weight subclass. However, the length of heparan sulfate chains was not changed by the metal. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled proteoglycans after heparitinase digestion showed that there were slightly more HSPG core proteins without a change of the size in lead-treated cell layer. It was, therefore, suggested that vascular endothelial cell layer after exposure to lead has more HSPG core proteins with fewer heparan sulfate chains without a change in length.

Blocking of TNF-alpha and IL-1 inhibits leukocyte infiltration at early, but not at late stage of S. aureus-induced arthritis and the concomitant cartilage destruction in rabbits.

We investigated the involvement of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the pathogenesis of heat-killed S. aureus-induced arthritis. TNF-alpha and IL-1beta peaked at 2 and 24 hr after the injection, respectively. Leukocyte infiltration within 12 hr of the inflammation was significantly inhibited (80%) by coinjection of anti-TNF-alpha mAb and IL-1 receptor antagonist (IL-1Ra) with S. aureus; however, leukocyte infiltration at 24 hr and thereafter was not inhibited by these agents. The loss of proteoglycan in S. aureus-induced arthritis was also unchanged either by anti-TNF-alpha mAb, IL-1Ra, or their combination. These results indicate that direct participation of TNF-alpha and IL-1 in the pathogenesis of S. aureus-induced arthritis may be limited to the early stage of inflammation and blocking of these cytokines did not result in diminishing the severity of inflammation. Thus, therapeutic approaches with the objective to suppress TNF-alpha and IL-1 may not be effective in the clinical treatment of gram-positive bacteria-induced arthritis.

Involvement of tumor necrosis factor-alpha, interleukin-1 beta, interleukin-8, and interleukin-1 receptor antagonist in acute lung injury caused by local Shwartzman reaction.

A local Shwartzman reaction (LSR) was prepared in rabbit lung as a model of acute lung injury. To induce LSR, intratracheal injection of lipopolysaccharide (LPS) 10 micrograms into the lower lobe of the right lung, followed 24 h later by i.v. injection of LPS (10 micrograms/kg). In the lung with the LSR, myeloperoxidase activity, representing neutrophil accumulation, peaked at 1-2 h and was sustained for 48 h after challenge with i.v. LPS. The lung water content peaked at 12 h, and decreased gradually. Histological findings showed diffuse interstitial widening, intra-alveolar leukocyte infiltration with hemorrhage, and alveolar exudate formation. The production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8), and IL-1 receptor antagonist (IL-1 Ra) in the lung was analyzed. TNF-alpha first elevated and peaked at 0.5 h (66.5 +/- 16.7 ng/g.lung), subsequently, IL-1 beta and IL-8 increased and peaked at 2 h (17.8 +/- 3.4 ng/g.lung and 336.9 +/- 49.6 ng/g.lung, respectively). IL-1Ra was present even before the challenge, and the production increased to show a dual peak (0.5 h, 1.5 +/- 0.2 micrograms/g.lung; and 2 h, 1.6 +/- 0.1 micrograms/g.lung), and a large concentration of IL-1Ra was sustained for 48 h. Immunohistochemistry showed that the cellular source of these cytokines was alveolar macrophages and infiltrating neutrophils. Thus, disclosing the kinetics of the generation of cytokines led to a better understanding of their roles, namely TNF-alpha as an initiator, IL-1 and IL-8 as amplifier and effector, and IL-1Ra as regulator of the intensity of acute inflammation.

Cadmium induces the production of high molecular weight heparan sulfate proteoglycan molecules in cultured vascular endothelial cells.

The purpose of the present study is to clarify whether or not cadmium-induced production of heparan sulfate in vascular endothelial cells includes: (1) an increase in the number of heparan sulfate proteoglycan (HSPG) molecules; (2) a formation of longer chains of heparan sulfate; and (3) a binding of more heparan sulfate chains to core proteins. Bovine aortic endothelial cells were cultured and metabolically labeled with [(3)H]glucosamine and [(35)S]sulfate in the presence of cadmium chloride. Radiolabeled HSPGs were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography and hydrodynamic size of HSPGs was characterized by gel filtration. Heparan sulfate chains were characterized by gel filtration after digestion with either papain or heparitinase. It was found that cadmium increases the incorporation of radioactive precursors into the high molecular weight subclass of HSPGs without a marked change of molecular weight of heparan sulfate chains (approximately 45 kDa). A sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [(35)S]methionine-labeled proteins after heparitinase digestion revealed that the endothelial cells actively produce a HSPG core with a high molecular weight (∼400 kDa), probably a perlecan core and the accumulation was increased by cadmium. HSPGs produced by cadmium-treated endothelial cells enhanced the [(3)H]thymidine incorporation in vascular smooth muscle cells cultured in the presence of basic fibroblast growth factor. It was therefore suggested that vascular endothelial cells after exposure to cadmium produce more perlecan molecules and this alteration may contribute to the antithrombogenic property of vascular wall and the formation of atherosclerosis after exposure to the metal through increase in anticoagulant heparan sulfate chains and stimulation of vascular smooth muscle proliferation, respectively.