Differential regulation of mast cell cytokines by both dexamethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580.

Activated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-alpha mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-alpha protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNF-alpha mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-alpha release but inhibited IL-4 release; notably, the ratio of TNF-alpha : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Ag-induced IL-4 and IL-6 mRNA more readily than TNF-alpha mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre- and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.

Benzylpenicillin differentially conjugates to IFN-gamma, TNF-alpha, IL-1beta, IL-4 and IL-13 but selectively reduces IFN-gamma activity.

It is known that beta-lactam antibiotics can conjugate to lysine and histidine residues on proteins via the carbonyl group of the opened beta-lactam ring. However, it is not known which proteins these drugs target and there is little work addressing whether conjugation is preferential for some proteins over others or if conjugation has functional consequences for the protein. We have previously shown that the beta-lactam antibiotic benzylpenicillin (BP) conjugates to IFN-gamma and reduces its activity. This interaction demonstrates selectivity, as BP does not bind to IL-4. Here, we extend our study to include other Th1 and Th2 cell-associated cytokines and two cytokines associated with inflammatory responses. We demonstrate by Western blotting that BP also conjugates to IL-1beta, IL-2, IL-5, IL-13 and TNF-alpha but not to IL-10. Densitometric analysis of leading cytokine bands on blots revealed that IFN-gamma always gave more intense BP-positive bands than any other cytokine analysed. Cytokines pre-incubated with BP at 37 degrees C in a protein-containing, serum-free medium were assayed for their biological activity. By in vitro bioassay, BP inhibited the ability of IFN-gamma but not IL-1beta or TNF-alpha to induce CD54 expression on epithelial cells. In addition, BP did not affect IL-4 or IL-13 inhibition of mast cell proliferation. When the pre-incubation temperature was reduced to 4 degrees C, BP did not conjugate to IFN-gamma or modulate its activity. BP retained its inhibitory effect on IFN-gamma activity when 20% FCS was added to the pre-incubation medium. In conclusion, BP conjugates to some cytokines but not others and this does not appear to be related to primary protein structure. Furthermore, of the cytokines studied, conjugation only to IFN-gamma is accompanied by inhibition of activity. This phenomenon is temperature dependent and occurs in the presence of serum. These findings provide further evidence for differential, direct drug-cytokine interactions. Such interactions may have therapeutic implications in terms of targeting cytokines to regulate their activity.

Nitric oxide: a regulator of mast cell activation and mast cell-mediated inflammation.

Nitric oxide (NO) plays diverse roles in physiological and pathological processes. During immune and inflammatory responses, for example in asthma, NO is generated at relatively high and sustained levels by the inducible form of nitric oxide synthase (NOS-2). NOS-2 derived NO regulates the function, growth, death and survival of many immune and inflammatory cell types. In the case of mast cells, NO suppresses antigen-induced degranulation, mediator release, and cytokine expression. The action of NO on mast cells is time dependent, requiring several hours, and noncGMP mediated, most probably involving chemical modification of proteins. NO inhibits a number of mast cell-dependent inflammatory processes in vivo, including histamine mediated vasodilatation, vasopermeation and leucocyte-endothelial cell attachment. In human asthma and animal models of lung inflammation the role of NO is harder to define. However, although there are conflicting data, the balance of evidence favours a predominantly protective role for NO. Mimicking or targeting NO dependent pathways may prove to be a valuable therapeutic approach to mast cell mediated diseases.

Characterization of specific IgE response in vitro against protein and drug allergens using atopic and normal donors.

BACKGROUND: As the incidence of allergy to different compounds increases in society, the need to understand and characterize specific IgE responses becomes obvious. Different cell culture systems have been evaluated for their ability to support such IgE secretion. METHODS: One system employed human peripheral lymphocytes (PBL) from normal donors stimulated with anti-CD3 activated T cells with or without the presence of allergens like benzylpenicillin (BP) and Phlenum pratense (PP). Secretion of IgE was analyzed in ELISA and compared to the IgG response to the nonallergenic antigen tetanus toxoid (TT). Another system employed stimulation of T and B cells with a heterotope, consisting of a T helper cell epitope derived from TT, and a B cell allergen epitope derived from BP. The specific IgE secretion was compared, using lymphocytes from normal as well as BP-allergic donors. RESULTS: Anti-CD3 stimulated T cells supported BP-specific IgE secretion in six of 11 normal donors. This response was inhibited in four donors and enhanced in two donors by the addition of the BP-allergen to the culture. In contrast, addition of the protein allergen (PP) or antigen (TT) to the same culture system inhibited both IgE and IgG synthesis in all experiments. Cells from the majority (10/16) of the BP-allergic donors failed to produce BP-specific IgE in vitro, when cultured in the presence of allergen. CONCLUSIONS: An allergen specific immune response is readily generated in vitro. The differential response against benzylpenicillin between different donor categories most probably reflects the level of pre-exposure to this allergen in vivo.

Penicillin conjugates to interferon-gamma and reduces its activity: a novel drug-cytokine interaction.

Beta-lactam antibiotics are the class of drug most frequently associated with IgE-mediated allergy but the mechanisms underlying this response are poorly understood. IFN-gamma is a key cytokine in immunity with regulatory actions on monocytes, NK cells, epithelial cells, and T and B lymphocytes. IFN-gamma promotes Th1 responses and inhibits Th2- and IgE-mediated responses. In this study we show, by Western blotting, that the prototype beta-lactam benzylpenicillin (BP) conjugates to human IFN-gamma but not to IL-4. The interaction of BP with IFN-gamma inhibited the cytokine's detection by immunoassay and impaired its activity, as assessed in three different assays: upregulation of MHC molecules on monocytes plus induction of nitric oxide synthesis and expression of monocyte chemoattractant protein-1 mRNA by epithelial cells. This is the first reported example of a direct drug-cytokine interaction and suggests a mechanism by which penicillin may disrupt IFN-gamma-dependent immune responses and promote allergy.

Nitric oxide in immunity and inflammation.

Nitric oxide (NO) is synthesised by many cell types involved in immunity and inflammation. The principal enzyme involved is the inducible type-2 isoform of nitric oxide synthase (NOS-2), which produces high-level sustained NO synthesis. NO is important as a toxic defense molecule against infectious organisms. It also regulates the functional activity, growth and death of many immune and inflammatory cell types including macrophages, T lymphocytes, antigen-presenting cells, mast cells, neutrophils and natural killer cells. However, the role of NO in nonspecific and specific immunity in vivo and in immunologically mediated diseases and inflammation is poorly understood. NO does not act through a receptor-its target cell specificity depends on its concentration, its chemical reactivity, the vicinity of target cells and the way that target cells are programmed to respond. At high concentrations as generated by NOS-2, NO is rapidly oxidised to reactive nitrogen oxide species (RNOS) that mediate most of the immunological effects of NOS-2-derived NO. RNOS can S-nitrosate thiols to modify key signalling molecules such as kinases and transcription factors. Several key enzymes in mitochondrial respiration are also inhibited by RNOS and this leads to a depletion of ATP and cellular energy. A combination of these interactions may explain the multiple actions of NO in the regulation of immune and inflammatory cells.

Phenotypic variation in mast cell responsiveness to the inhibitory action of nitric oxide.

OBJECTIVE AND DESIGN: The aim of this study was to investigate the possible phenotypic variations between mast cells in terms of their responsiveness to the inhibitory actions of nitric oxide. MATERIALS: Unfractionated mouse peritoneal cells, purified rat peritoneal mast cells, mouse bone marrow-derived mast cells of the C1.MC/C57.1 line (cultured mouse mast cells, CMMC) and rat basophilic leukemia cells of the RBL-2H3 line were used. METHODS: Mast cells were cultured with interferon-gamma (IFN-gamma)-stimulated mouse peritoneal cells as a source of nitric oxide, or with the nitric oxide donor S-nitrosoglutathione (SNOG). After 24 h culture, the mast cells were challenged with anti-IgE, antigen, or calcium ionophore A23187, and degranulation measured as release of [3H]serotonin. RESULTS: Addition of IFN-gamma to mouse peritoneal cells led to nitric oxide synthesis and this was associated with decreased IgE-mediated mast cell degranulation. IFN-gamma did not induce nitric oxide production by CMMC and degranulation of CMMC was not inhibited by nitric oxide generated by co-cultured IFN-gamma-activated peritoneal cells. The nitric oxide donor SNOG inhibited degranulation of purified rat peritoneal mast cells, but not RBL-2H3 cells, stimulated by either IgE cross-linking or calcium ionophore. CONCLUSIONS: The inhibitory effects of nitric oxide on mast cell degranulation are variable and selective for phenotype. Such phenotypic differences may reflect important variations in regulation of mast cell function.

Presentation of IFN-gamma to nitric oxide-producing cells: a novel function for mast cells.

We report that mast cells can bind and present IFN-gamma in a functionally active form to macrophages. Flow-cytometric analysis revealed that biotinylated IFN-gamma bound equally well to purified peritoneal mast cells from both IFN-gammaR knockout and wild-type mice, indicating a non-IFN-gammaR binding site. Purified peritoneal mast cells, loaded with IFN-gamma for 30 min and washed, were able to induce NO synthesis by peritoneal macrophages. This response required cell contact and expression of IFN-gammaR on the responding macrophages, but not the mast cells. Human HMC-1 mast cells were also able to present IFN-gamma to mouse macrophages. Enzyme treatment of mouse mast cells revealed that binding of IFN-gamma was predominantly to chondroitin sulfate B (dermatan sulfate). Binding of IFN-gamma to dermatan sulfate was confirmed by inhibition ELISA. This study demonstrates for the first time that mast cells can present IFN-gamma to other cells via glycosaminoglycans. Mast cells may act as a reservoir of surface-stored functionally active cytokines.

Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation.

The beta chemokine eotaxin is a potent eosinophil activator and chemoattractant. We examined immunohistochemically eotaxin protein expression in a range of normal rat tissues and in rat lung during Sephadex particle-induced pulmonary inflammation. The time course of eotaxin expression in lung at various time points after Sephadex administration was related to the appearance of eosinophils in the bronchoalveolar lavage fluid and tissue distribution of eotaxin receptor (CCR3) positive cells. Results showed that eotaxin protein was constitutively expressed by both lung airway epithelial cells and gut epithelial cells in normal tissues in the absence of inflammation. During Sephadex induced pulmonary inflammation, eotaxin expression increased in alveolar macrophages prior to the major increase in eosinophil numbers which reached a peak at 72 h. The pattern of eotaxin pulmonary expression and the location of CCR3 receptor positive cells suggest a chemoattractant gradient resulting in migration firstly into the tissue and subsequently through the airway epithelium into the airways. Treatment of rats with the glucocorticoid dexamethasone or the immunosuppressant cyclosporin A reduced eosinophil entry into lung tissue and airways but had no apparent effect on eotaxin expression in vivo, indicating that both these drugs inhibit eosinophil recruitment either by an eotaxin-independent mechanism, or by targetting factors that synergise with eotaxin, or an event post eotaxin expression.

Reciprocal effects of interleukin-4 and interferon-gamma on immunoglobulin E-mediated mast cell degranulation: a role for nitric oxide but not peroxynitrite or cyclic guanosine monophosphate.

We report that cultured rat peritoneal cells spontaneously synthesize nitric oxide and this is associated with active suppression of mast cell secretory function. Addition of interleukin-4 (IL-4) or the nitric oxide synthase inhibitor N-monomethyl-l-arginine to peritoneal cells inhibited nitric oxide synthesis and enhanced anti-IgE-mediated mast cell degranulation, measured as serotonin release. Interferon-gamma (IFN-gamma) completely overcame the enhancement of serotonin release and suppression of nitrite production induced by IL-4. Over several experiments, with or without IL-4 and/or IFN-gamma, serotonin release correlated inversely with nitrite production. On a cell-for-cell basis, non-mast cells produced approximately 30 times more nitrite than mast cells in peritoneal cell populations, with or without IFN-gamma stimulation. The nitric oxide donor S-nitrosoglutathione inhibited anti-IgE-induced serotonin release from purified mast cells, whereas 8-bromo-cyclic GMP, the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, superoxide dismutase and the peroxynitrite scavenger uric acid, were without effect. We conclude that IL-4 and IFN-gamma reciprocally regulate mast cell secretory responsiveness via control of nitric oxide synthesis by accessory cells; the nitric oxide effect on mast cells is direct but does not involve cyclic GMP or peroxynitrite.

Poliomyelitis in intraspinally inoculated poliovirus receptor transgenic mice.

Mice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice.

Direct detection of Sabin poliovirus vaccine strains in stool specimens of first-dose vaccinees by a sensitive reverse transcription-PCR method.

A multiplex reverse transcription-PCR method was optimized to monitor the duration of excretion of Sabin poliovirus strains in stools of vaccinees following administration of the first dose of the trivalent oral vaccine. The assay detected approximately 1 50% tissue culture infective dose of each poliovirus serotype spiked into cell culture media. Although PCR inhibitors were frequently encountered in the stool specimens, a 1:20 dilution of the extracted RNA was sufficient to obtain a positive PCR result. Analysis of 195 stool specimens collected from 26 vaccinees showed that poliovirus types 1, 2, and 3 were identified more frequently by PCR than by tissue culture isolation. The percentages of specimens positive by PCR for poliovirus types 1, 2, and 3 were 67.2, 82.6, and 53.8, respectively. In contrast, the culture method identified types 1, 2, and 3 virus in 55.4, 64.1, and 27.7% of the samples, respectively. Poliovirus type 2 excretion was detected by PCR in practically all of the oral poliovirus vaccine recipients for 4 to 8 weeks following vaccination. In contrast, excretion of type 1 and 3 viruses was more variable, with a range of 1 to 8 weeks. Shedding of type 3 virus ceased in approximately 70% of vaccinees within a week after immunization. In addition to an enhanced sensitivity for the detection of poliovirus, this PCR method permits the direct characterization of virus in stool specimens without further passage in culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Characterization of mouse lines transgenic with the human poliovirus receptor gene.

Two mouse lines transgenic with the human poliovirus receptor gene (PVR), TGM-PRG-1 and TGM-PRG-3, were characterized to determine whether transgene copy number and PVR expression levels influence susceptibility to poliovirus. The mouse lines have been bred for more than 10 generations and the transgene was stably transmitted to progeny as determined by Southern blot hybridization and restriction fragment length polymorphism. The transgene copy number is 10 in the TGM-PRG-3 mouse line and one in the TGM-PRG-1 mouse line. Abundance of PVR RNA is on average three-fold higher in TGM-PRG-3 relative to TGM-PRG-1 tissues, and the abundance of the receptor molecule is three-fold higher in TGM-PRG-3 central nervous system tissues compared to TGM-PRG-1 tissues as determined by Western blot analysis. When TGM-PRG-1 and TGM-PRG-3 mice were inoculated intracranially with a neurovirulent type III poliovirus strain, they developed clinical symptoms and CNS lesions characteristic of human poliomyelitis. These results indicate that the PVR gene is expressed as a functional receptor in the CNS of both mouse lines rendering the mice susceptible to poliovirus infection. Even though the two mouse lines have different copy numbers of the transgene and different levels of PVR RNA and protein, they are similar in their susceptibility to poliovirus.

Tumour necrosis factor-alpha expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A.

1. Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-alpha is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-alpha expression influences recruitment of specific inflammatory cell types. 2. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24-72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3. Messenger RNA encoding TNF-alpha was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-alpha protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. 4. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-alpha mRNA expression by BAL cells. 5. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-alpha, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-alpha expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.

Nitric oxide inhibits IgE-mediated degranulation of mast cells and is the principal intermediate in IFN-gamma-induced suppression of exocytosis.

IFN-gamma regulates various aspects of rodent peritoneal mast cell function, including mediator release, cell growth, TNF-alpha-mediated cytotoxicity, and MHC class II expression. We investigated whether the suppressive action of IFN-gamma on IgE/Ag-mediated degranulation of mast cells is mediated via synthesis of nitric oxide. Incubation of mouse peritoneal cells with L-NMMA, an inhibitor of nitric oxide synthase, or in medium lacking the nitric oxide precursor L-arginine reversed the inhibitory effect of IFN-gamma on Ag-induced serotonin release. Furthermore, the nitric oxide donors sodium nitroprusside and S-nitrosoglutathione inhibited degranulation, and this effect was direct, since it was seen equally on purified and unfractionated mast cells and occurred independently of IFN-gammaR expression. Additional experiments revealed that accessory cells in peritoneal cell populations were the principal target for the action of IFN-gamma and the main source of nitric oxide; the cytokine was more potent on unfractionated compared with purified mast cells, and IFN-gamma induced detectable nitrite production in mixed peritoneal cells, but not in purified mast cells. These studies show that IFN-gamma induces nitric oxide production in peritoneal cell populations, and that synthesized nitric oxide directly inhibits the IgE-mediated secretory function of mast cells. The activation of nitric oxide-producing cells in the tissue microenvironment may be important in the control of mast cell-dependent allergic reactions.