Reduced adrenal response to bacterial lipopolysaccharide in interleukin-6-deficient mice.

Administration of bacterial lipopolysaccharide (LPS) in rodents induces the release of pro-inflammatory cytokines [tumor necrosis factor (TNF), interleukin (IL)-1, IL-6] and of ACTH and corticosterone. IL-6 is probably an important cytokine in the interaction between the immune system and the hypothalamus-pituitary-adrenal (HPA) axis, but so far the role of IL-6 in lipopolysaccharide (LPS)-induced HPA activation has not been established unequivocally. We examined the effects of intraperitoneal administration of LPS (range 0.25-2000 pg/mouse) on plasma corticosterone, TNFalpha and IL-1alpha levels in IL-6-deficient (IL-6 -/-) and wildtype control (IL-6 +/+) mice. Plasma corticosterone levels increased within one hour in both mouse strains. The corticosterone response was significantly reduced in IL-6 -/- mice, but no differences in TNFalpha or in IL-1alpha plasma levels were found between the two strains. Next, we studied the involvement of IL-1alpha or TNFalpha in the responses to LPS in IL-6 -/- and IL-6 +/+ mice by infusion of recombinant human IL-1 receptor antagonist (IL-1ra), or by injection of anti-TNFalpha antibodies. Pretreatment with IL-1ra or with anti-TNFalpha did not affect the corticosterone response to LPS, neither in IL-6 -/-, nor in IL-6 +/+ mice. Our data suggest that in the stimulation of the HPA axis by LPS in mice blockade of either IL-1alpha or TNFalpha may be compensated for by other mediators. The reduced adrenal response after LPS administration found in IL-6 -/- mice indicates a distinct role for IL-6 in the activation of the HPA axis by LPS.

Reduced adrenal response and increased mortality after systemic Klebsiella pneumoniae infection in interleukin-6-deficient mice.

During bacterial infections, both the immune system and the hypothalamus-pituitary-adrenal (HPA) axis are activated. The role of IL-6 in the activation of the HPA axis during bacterial sepsis is not fully understood. The aim of the present study was to investigate the role of endogenous IL-6 in a potentially lethal infection with Klebsiella pneumoniae and the concomitant activation of the HPA axis. We examined the mortality of IL-6-/- and IL-6+/+ mice after intravenous (i.v.) infection with K. pneumoniae as well as the bacterial outgrowth in several organs. Subsequently, the influence of endogenous IL-6 on the effect of i.v. administration of K. pneumoniae on the plasma levels of corticosterone and the pro-inflammatory cytokines TNF-alpha and IL-1alpha was investigated in these mice. The present study demonstrates that IL-6-/- mice are more susceptible than IL-6+/+ mice to a systemic Gram-negative infection with K. pneumoniae, leading to increased outgrowth of microorganisms in the organs of the mice. Moreover, this infection is associated with a reduced adrenal response in IL-6-/- mice. We conclude that IL-6-/- mice are more susceptible to Gram-negative bacterial infections, which is mainly due to an impaired recruitment of granulocytes to the site of infection in the absence of IL-6. Furthermore, the reduced adrenal response may be an explanation for the strong inflammatory response with higher TNF-alpha plasma levels in IL-6-/- mice.

Oreochromis mossambicus (tilapia) corticotropin-releasing hormone: cDNA sequence and bioactivity.

Although hypothalamic corticotropin-releasing hormone (CRH) is involved in the stress response in all vertebrate groups, only a limited number of studies on this neuroendocrine peptide deals with non-mammalian neuroendocrine systems. We determined the cDNA sequence of the CRH precursor of the teleost Oreochromis mossambicus (tilapia) and studied the biological potency of the CRH peptide in a homologous teleost bioassay. Polymerase chain reaction (PCR) with degenerate and specific primers yielded fragments of tilapia CRH cDNA. Full-length CRH cDNA (988 nucleotides) was obtained by screening a tilapia hypothalamus cDNA library with the tilapia CRH PCR products. The precursor sequence (167 amino acids) contains a signal peptide, the CRH peptide and a motif conserved among all vertebrate CRH precursors. Tilapia CRH (41 aa) displays between 63% and 80% amino acid sequence identity to CRH from other vertebrates, whereas the degree of identity to members of the urotensin I/urocortin lineage is considerably lower. In a phylogenetic tree, based on alignment of all full CRH peptide precursors presently known, the three teleost CRH precursors (tilapia; sockeye salmon, Oncorhynchus nerka; white sucker, Catostomus commersoni) form a monophyletic group distinct from amphibian and mammalian precursors. Despite the differences between the primary structures of tilapia and rat CRH, maximally effective concentrations of tilapia and rat CRH were equally potent in stimulating adrenocorticotropic hormone (ACTH) and alpha-MSH release by tilapia pituitaries in vitro. The tilapia and salmon CRH sequences show that more variation exists between orthologous vertebrate CRH structures, and teleost CRHs in particular than previously recognized. Whether the structural differences reflect different mechanisms of action of this peptide in the stress response remains to be investigated.

Increased susceptibility to systemic candidiasis in interleukin-6 deficient mice.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates multiple aspects of the innate immune response. It has been recently shown that endogenous IL-6 is crucial for an efficient defence against severe infections with Gram-negative and Gram-positive bacteria. The aim of the present study was to investigate the role of endogenous IL-6 in the defence against infection with the yeast Candida albicans. During experimental candidemia, IL-6 deficient mice (IL-6-/-) had a decreased survival and an increased fungal load in their organs when compared with IL-6+/+ controls, despite increased plasma concentrations of tumour necrosis factor-alpha (TNF), interleukin-1 alpha (IL-1 alpha) and IL-1 beta, IL-6-/- mice were not able to mount an efficient neutrophil response during the infection. When mice were rendered neutropenic by cyclophosphamide, neutropenic IL-6-/- mice were equally susceptible to C. albicans when compared to neutropenic IL-6+/+ mice, implying that neutrophils mediate the beneficial effect of endogenous IL-6. In conclusion, IL-6-/- mice are more susceptible to disseminated candidiasis, and the effect of IL-6 is most likely mediated by neutrophils.

Reduced cartilage proteoglycan loss during zymosan-induced gonarthritis in NOS2-deficient mice and in anti-interleukin-1-treated wild-type mice with unabated joint inflammation.

OBJECTIVE: To investigate the role of nitric oxide (NO) and interleukin-1 in (IL-1) joint inflammation and cartilage destruction during zymosan-induced gonarthritis (ZIA). METHODS: Monarticular arthritis was elicited by intraarticular injection of zymosan. The effect of NO deficiency on arthritis was studied in mice with genetically disrupted NOS2. The role of IL-1 was examined by treating wild-type mice with neutralizing anti-murine IL-1(alpha+beta) antibodies. Joint swelling was measured externally by the increased uptake of circulating 99mtechnetium pertechnetate. Proteoglycan (PG) synthesis was assessed using 35S-sulfate incorporation into patellae ex vivo. Histology evaluated exudation and infiltration of leukocytes and the extent of cartilage destruction. RESULTS: The proinflammatory mediators NO, IL-1, and IL-6 were released by the articular tissues during the first hours of inflammation. Interestingly, anti-IL-1 treatment moderately reduced, and NOS2 deficiency moderately enhanced, joint swelling. However, the influx of neutrophils into the joint occurred independently of IL-1 and NOS2 activities. In the first week of inflammation, chondrocyte PG synthesis was significantly suppressed and chondrocytes became unresponsive to their essential anabolic factor, insulin-like growth factor 1 (IGF-1). Anti-IL-1 treatment or NOS2 deficiency prevented the inhibition of PG synthesis, and the chondrocytes remained IGF-1 responsive. Intraarticular injections of IL-1alpha into NOS2-deficient mice did not affect PG synthesis, thus proving that NO mediated this IL-1 effect in vivo. Furthermore, histology showed that cartilage PG loss was markedly ameliorated in NOS2-deficient and anti-IL-1-treated mice. Intermediate cartilage pathology was found in mice that were heterozygous for disrupted NOS2. CONCLUSION: IL-1 and NO play a minor role in edema and neutrophil influx, but a major role in cartilage destruction of ZIA. In this model of murine arthritis, cartilage destruction was, for the most part, caused by pronounced suppression of PG synthesis and IGF-1 unresponsiveness of the chondrocytes, which were induced by de novo-synthesized IL-1 and were mediated by NOS2 activation.

Interleukin-6 reduces cartilage destruction during experimental arthritis. A study in interleukin-6-deficient mice.

Using interleukin (IL)-6-deficient (IL-6(0/0) mice or wild-type mice, we investigated the controversial role of IL-6 in joint inflammation and cartilage pathology during zymosan-induced arthritis (ZIA). Monoarticular arthritis was elicited by injection of zymosan into the right knee joint cavity. Production of IL-1, tumor necrosis factor (TNF), IL-6, and nitric oxide by the inflamed knee was assessed in washouts of joint capsule specimens. Plasma corticosterone was measured using a radioimmunoassay. Proteoglycan synthesis was assessed using [35S]sulfate incorporation into patellas ex vivo. Joint swelling was quantified by joint uptake of circulating 99mTechnetium pertechnetate. Histology was taken to evaluate cellular infiltration and cartilage damage. Zymosan caused a rapid increase in articular IL-1, IL-6, TNF, and NO levels. Except for IL-6, the released amounts and time course of these mediators were comparable in the IL-6-deficient mice and the wild-type mice. Elevated plasma corticosterone levels were measured during the first day of arthritis in both strains. At day 2 of ZIA, joint inflammation (joint swelling and cell exudate) in IL-6-deficient mice was comparable with that in the wild-type mice. The marked suppression of chondrocyte proteoglycan synthesis and proteoglycan degradation were on the average higher in the IL-6-deficient mice. Together this resulted in a more pronounced proteoglycan depletion in the IL-6-deficient mice as compared with the wild-type mice during the first week of arthritis. Injection of recombinant IL-6 into the joint cavity corrected the IL-6 deficiency and significantly reduced cartilage destruction. Inflammation was more chronic in the wild-type mice, and these mice also showed a higher prevalence for osteophyte formation. In ZIA, IL-6 plays a dual role in connective tissue pathology, reducing proteoglycan loss in the acute phase and enhancing osteophyte formation in the chronic phase. The latter could be related to the more severe joint inflammation as seen in the normal (IL-6-producing) animals during the chronic phase of arthritis.

Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes.

A total of 13 new Giardia isolates were established in axenic culture. All of the new isolates were obtained by excystation of Giardia cysts from the feces of patients in Dutch hospitals. These isolates were subjected to isoenzyme and DNA analysis together with isolates from Poland, Belgium, and various other parts of the world. Isoenzyme analysis revealed that nearly all of the newly established isolates exhibited unique zymodemes. Isolates obtained from individuals from Belgium and Poland, on the other hand, displayed single zymodemes. Genomic DNA libraries were constructed from isolates belonging to the latter two zymodemes; specific and common recombinant DNA clones were selected from these libraries. Differential screening revealed that the two isolates had only 80% of the clones in common. Restriction-fragment-length polymorphism analysis using three different probes together with two synthetic probes that are complementary to Giardia structural protein genes led to the separation of all isolates into two major groups; within these groups, a further division could be made by application of other techniques or probes. The results of DNA analysis and zymodeme classification were in general agreement; in the present report they are compared with the data in the literature and discussed.