Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis and the other Bordetella species produce a novel adenylate cyclase toxin which enters target cells to catalyze the production of supraphysiologic levels of intracellular cyclic adenosine monophosphate (cAMP). In these studies, dialyzed extracts from B. pertussis containing the adenylate cyclase toxin, a partially purified preparation of adenylate cyclase toxin, and extracts from transposon Tn5 mutants of B. pertussis lacking the adenylate cyclase toxin, were used to assess the effects of adenylate cyclase toxin on human peripheral blood monocyte activities. Luminol-enhanced chemiluminescence of monocytes stimulated with opsonized zymosan was inhibited greater than 96% by exposure to adenylate cyclase toxin-containing extract, but not by extracts from adenylate cyclase toxin-deficient mutants. The chemiluminescence responses to particulate (opsonized zymosan, Leishmania donovani, and Staphylococcus aureus) and soluble (phorbol myristate acetate) stimuli were inhibited equivalently. The superoxide anion generation elicited by opsonized zymosan was inhibited 92% whereas that produced by phorbol myristate acetate was inhibited only 32% by B. pertussis extract. Inhibition of oxidative activity was associated with a greater than 500-fold increase in monocyte cAMP levels, but treated monocytes remained viable as assessed by their ability to exclude trypan blue and continued to ingest particulate stimuli. The major role of the adenylate cyclase toxin in the inhibition of monocyte oxidative responses was demonstrated by: 1) little or no inhibition by extracts from B. pertussis mutants lacking adenylate cyclase toxin; 2) high level inhibition with extract from B. parapertussis, a related species lacking pertussis toxin; and 3) a reciprocal relationship between monocyte cAMP levels and inhibition of opsonized zymosan-induced chemiluminescence using both crude extract and partially purified adenylate cyclase toxin. Pertussis toxin, which has been shown to inhibit phagocyte responses to some stimuli by a cAMP-independent mechanism, had only a small (less than 20%) inhibitory effect when added at concentrations up to 100-fold in excess of those present in B. pertussis extract. These data provide strong support for the hypothesis that B. pertussis adenylate cyclase toxin can increase cAMP levels in monocytes without compromising target cell viability or impairing ingestion of particles and that the resultant accumulated cAMP is responsible for the inhibition of oxidative responses to a variety of stimuli.     

The antibody responses to myelin basic protein (BP) in Lewis rats: the effects of Bordetella pertussis.

A time-course study was made of the systemic humoral immune response of Lewis rats to myelin basic protein (BP) as influenced by the dosage of ancillary pertussis adjuvant. Peak activities were observed 5 to 7 weeks after injection. When injected proximal to BP and Mycobacterium butyricum in complete Freund's adjuvant (CFA), Bordetella pertussis at the level of 4 billion organisms doubled the antibody-binding activity of rat sera for 125I-labeled BP as compared to activities obtained with 0, 2, 6, or 8 billion. The severity of clinical symptoms of experimental allergic encephalomyelitis (EAE) at the end of the 2nd week was greatest in rats receiving 64 billion organisms, the very same rats that displayed a severely dampened humoral immune response to BP 5 weeks later. When pertussis was injected i.p. rather than proximal to the CFA mixture, the time-course of the humoral immune response displayed a different profile--unusually high binding activities at the time of onset of EAE that fluctuated back and forth from high to low and that eventually dampened to an intermediate level.     

Selective inhibition of COX-2 is beneficial to mice infected intranasally with VSV

Cyclooxygenase (COX) is the key enzyme for prostaglandin (PG) synthesis. PGs are mediators of many critical physiological and inflammatory responses. There are two isoforms, COX-1 and COX-2, both of which are constitutively expressed in the central nervous system (CNS). Studies have shown that COX-1 and COX-2 are involved in physiological and pathological conditions of the brain. However, little is known about the role(s) of COX in the host defense system against a viral infection in the CNS. In this report, we used Vesicular Stomatitis Virus (VSV) induced acute encephalitis to distinguish between the contribution(s) of the two isoforms. COX-2 activity was inhibited with a COX-2 selective drug, celecoxib (Celebrex), and COX-1 was antagonized with SC560. We found that inhibition of COX-2 led to decreased viral titers, while COX-1 antagonism did not have the same effect at day 1 post infection. 5-lipooxygenase (5-LO) expression and neutrophil recruitment in the CNS were increased in celecoxib-inhibited mice. Furthermore, mice treated with celecoxib expressed more Nitric Oxide Synthase-1 (NOS-1), a crucial component of the innate immune system in the restriction of VSV propagation. The expression of type 1 cytokines, IFN-gamma and IL-12, were also increased in celecoxib-treated mice.     

Regulation of inflammatory responses by oncostatin M

Oncostatin M (OM) is a pleiotropic cytokine produced late in the activation cycle of T cells and macrophages. In vitro it shares properties with related proteins of the IL-6 family of cytokines; however, its in vivo properties and physiological function are as yet ill defined. We show that administration of OM inhibited bacterial LPS-induced production of TNF-alpha and lethality in a dose-dependent manner. Consistent with these findings, OM potently suppressed inflammation and tissue destruction in murine models of rheumatoid arthritis and multiple sclerosis. T cell function and Ab production were not impaired by OM treatment. Taken together these data indicate the activities of this cytokine in vivo are antiinflammatory without concordant immunosuppression.     

Altered inflammatory responses in TLR5-deficient mice infected with Legionella pneumophila

Legionella pneumophila (Lp), an important cause of morbidity and mortality from pneumonia, infects alveolar macrophages (AMs) and is recognized by several TLRs as well as Birc1e (NAIP5) and IL-1 converting enzyme-protease activating factor. We examined the role of TLR5 during the murine response to aerosolized Lp infection. At 4 h after infection, Tlr5(-/-) mice had lower numbers of polymorphonuclear neutrophils (PMNs) in their broncho-alveolar lavage fluid in comparison to wild-type (WT) mice. At 24 and 72 h, the PMN recruitment was similar. WT mice infected with a flagellin-deficient strain (LpFlaA-) also showed an impaired early PMN response at 4 h compared with those infected with the WT strain. There was no consistent difference in bacterial counts at any of the time points when comparing the Tlr5(-/-) and WT mice. However, at 6 days after infection, the Tlr5(-/-) mice had increased leukocytic infiltrates in the alveolar and peribronchial interstitial spaces that were consistent with organizing pneumonia. We also examined the role of TLR5 during macrophage infection. In contrast to bone marrow-derived macrophages, AMs secreted TNF-alpha after stimulation with purified flagellin. In addition, WT, but not Tlr5(-/-), AMs produced TNF-alpha after stimulation with Lp. Live LpFlaA- did not induce TNF-alpha secretion in AM. These results suggested that AMs recognize Lp flagellin and that a majority of the Lp-induced TNF-alpha response is TLR5-mediated. Thus, TLR5 mediates recognition of Lp in AMs and performs a distinct role during the in vivo pulmonary immune response through regulation of early PMN recruitment and subsequent later development of pneumonia.     

Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses

Recombinant Norwalk virus-like particles (rNV VLPs) were administered to BALB/c mice by the intranasal (i.n.) route to evaluate the induction of mucosal antibody responses. The results were compared to systemic and mucosal responses observed in new and previous studies (J. M. Ball, M. E. Hardy, R. L. Atmar, M. E. Connor, and M. K. Estes, J. Virol. 72:1345-1353, 1998) after oral administration of rNV VLPs. Immunizations were given in the presence or absence of a mucosal adjuvant, mutant Escherichia coli heat-labile toxin LT(R192G). rNV-specific immunoglobulin G (IgG) and fecal IgA were evaluated by enzyme-linked immunosorbent assay. The i.n. delivery of rNV VLPs was more effective than the oral route at inducing serum IgG and fecal IgA responses to low doses of rNV particles. Vaginal responses of female mice given VLPs by the i.n. and oral routes were also examined. All mice that received two immunizations with low doses i.n. (10 or 25 microg) of rNV VLPs and the majority of mice that received two high doses orally (200 microg) in the absence of adjuvant had rNV-specific serum IgG, fecal, and vaginal responses. Additional experiments evaluated whether rNV VLPs can function as a mucosal adjuvant by evaluating the immune responses to two soluble proteins, keyhole limpet hemocyanin and chicken egg albumin. Under the conditions tested, rNV VLPs did not enhance the serum IgG or fecal IgA response to these soluble proteins when coadministered by the i.n. or oral route. Low doses of nonreplicating rNV VLPs are immunogenic when administered i.n. in the absence of adjuvant, and addition of adjuvant enhanced the magnitude and duration of these responses. Recombinant NV VLPs represent a candidate mucosal vaccine for NV infections in humans.     

Detection of the gene sequences of the leukocytosis (lymphocytosis)-stimulating factor of Bordetella pertussis in Bordetella parapertussis and Bordetella bronchiseptica by using molecular hybridization

The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3) chromosome DNA carrying the pertussis toxin (PT) operon was cloned on vector plasmid pUC19 in Escherichia coli. Three fragments (1.14 Kb KpnI-PstI, 1.27 Kb PstI-PstI, and 0.96 Kb PstI-PstI) were obtained from the resulting hybrid plasmid, coded pRH119, by electrophoretic techniques and used as a combined molecular probe for analysis of the EcoRI-digested and PstI-digested chromosomal DNA of B. pertussis strain 475 in phase I, B. pertussis in phase IV, B. parapertussis strains 504 and 17903, B. bronchiseptica strain 214, and B. parapertussis strain 17903 (a convertant obtained by means of B. pertussis phage 134), as well as B. pertussis phage 134. Southern blot hybridization under the conditions of 100% DNA-DNA homology showed the presence of DNA sequences characteristic of the PT operon in all cases except the DNA of phage 134; moreover, the use of the above-mentioned probe made it possible to hybridize all EcoRI-fragments of chromosomal DNA, having the same molecular size (4.7 Kb). Consequently, the PT genes in the above Bordetella species were mapped in identical loci.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of defence responses in avocado roots infected with Phytophthora cinnamomi (Rands)

Phytophthora cinnamomi occurs worldwide and has a host range in excess of 1,000 plant species. Avocados (Persea americana Mill) have been described as highly susceptible to this soil-borne pathogen. Here, the regulation of defence responses in avocado root seedlings inoculated with P. cinnamomi mycelia is described. A burst of reactive oxygen species (ROS) was observed 4 days after inoculation. The higher physiological concentration of H₂O₂ induced by P. cinnamomi on avocado roots had no effect on in vitro growth of the oomycete. Total phenols and epicathecin content showed a significant decrease, but lignin and pyocianidins exhibited no changes after inoculation. Also, increased nitric oxide (NO) production was observed 72 h after treatment. We studied the effects of one NO donor [sodium nitroprusside (SNP)], and one NO scavenger [2- to 4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (CPTIO)] to determine the role of NO during root colonisation by P. cinnamomi mycelia. Pretreatment of the roots with CPTIO, but not with SNP, inhibited root colonisation suggesting an important role for NO production during the avocado–P. cinnamomi interaction. Our data suggest that although defence responses are activated in avocado roots in response to P. cinnamomi infection, these are not sufficient to avoid pathogen invasion.     

Proliferation of spleen cells in response to Bordetella pertussis

Vaccine strain 305 of B. pertussis in a dose of 10(8)-10(11) cells was shown to be mitogenic for splenocytes of BALB/c mice and nude mice. When added in a dose of 10(10) B. pertussis exerted a more pronounced mitogenic effect than phytohemagglutinin P, which was less powerful, however, than that of Con A, B. pertussis caused a greater stimulation of DNA synthesis in lymphocytes than B mitogens whose action depended on the differentiation stages of B lymphocytes. This is likely to hint towards a possible action of B. pertussis on immature B cells and/or their precursors. The cells of T lineage (T1 cells and/or T precursors) can also be involved.