Substance P-induced IL-12 production by murine macrophages.

Previous investigations in our laboratory have suggested that substance P (NK-1) receptor expression by macrophages contributes to the resistance against the intracellular bacterial pathogen, Salmonella. To investigate possible mechanisms for such resistance, macrophages were cultured with varying concentrations of a substance P agonist to investigate the ability of this neuropeptide to augment IL-12 expression. The substance P agonist was a potent inducer of both IL-12p35 and IL-12p40 mRNA expression in cultured macrophages. The kinetics of this response were maximal within 6 h and could be observed with concentrations of substance P agonist as low as 0.1 nM. The nonpeptide, substance P receptor antagonist, CP96-345, significantly blocked agonist-induced IL-12 mRNA expression, further demonstrating that this effect was mediated through an NK-1 receptor. Substance P agonist alone could stimulate substantial secretion of IL-12p40, but not IL-12p70, by cultured macrophages. Thus, the substance P agonist had the ability to augment IL-12p35 and IL-12p40 mRNA expression, but not to increase IL-12p70 secretion. Like IFN-gamma, we found that substance P could combine with LPS to significantly augment the secretion of bioactive IL-12p70. The costimulatory effects of substance P agonist plus LPS on IL-12 mRNA expression were additive; however, this combination resulted in synergistic secretion of IL-12p70 by macrophages. Together, these results demonstrate the ability of NK-1 receptors to signal IL-12 production by macrophages and suggest mechanisms for substance P-induced modulation of cellular immunity.

Reverse transcriptase-polymerase chain reaction amplification and partial sequence of T helper 1- and T helper 2-type lymphokine genes from the owl monkey (Aotus trivirgatus).

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify selected lymphokine mRNAs from phytohemagglutinin-activated leukocytes of the owl monkey (Aotus trivirgatus). Interleukin-2 (IL-2), IL-4, IL-13, and interferon-gamma were selected as lymphokine mRNAs of interest, since expression of these cytokines helps define the type of T helper lymphocyte response (i.e., TH1 versus TH2). Because sequences for these lymphokine genes were not available for the owl monkey, multiple PCR primers for each lymphokine gene were designed based on published human sequences. Various PCR primer pairs were then used in the RT-PCR to determine the conditions for optimal amplification of each owl monkey cytokine mRNA. In addition, each PCR primer pair was compared for the ability to amplify lymphokine mRNAs from other primate species, including African green (Cercopithecus aethiops), squirrel (Saimiri sciureus), and rhesus (Macaca mulatta) monkeys. The specificity and sensitivity of optimal primer pair was also demonstrated by amplification of as little as 10 fg of each lymphokine gene in a background of 300 ng of irrelevant cDNA. Finally, partial sequences of owl monkey coding regions for IL-2, IL-13, and interferon-gamma were determined and compared for homology with their human counterparts. Together, these studies define specific and sensitive conditions for detection of lymphokine mRNA expression in the owl monkey and provide partial sequence information of the coding region for these lymphokines. This investigation should provide molecular probes to investigate the immune response against malaria and the effectiveness of malaria vaccines in the owl monkey that models this human disease.

Increased susceptibility of mice to Salmonella infection following in vivo treatment with the substance P antagonist, spantide II.

Successful resolution of salmonellosis in naive mice depends in large part upon IL-12-induced IFN-gamma production to eliminate this intracellular pathogen of macrophages. In the present study we questioned the contribution that expression of substance P receptors makes to the protective response following oral inoculation with a lethal dose of Salmonella. Such a relationship was suggested when oral inoculation with Salmonella induced rapid and dramatic increases in substance P receptor mRNA expression within Peyer's patches and mesenteric lymph nodes and subsequently in the spleen. The importance of substance P receptor expression in vivo was further suggested by pretreatment of mice with the substance P antagonist, spantide II, before oral inoculation with Salmonella. Mice pretreated with spantide II and then orally inoculated developed advanced salmonellosis and had significantly reduced survival rates compared with mice pretreated with a control peptide. Treatment with spantide II significantly reduced early Salmonella-induced IL-12p4O and IFN-gamma mRNA expression at mucosal sites, suggesting a mechanism for the reduced ability of spantide II-treated mice to resist this pathogen. Increased susceptibility to salmonellosis was not due to 1) spantide II-induced alterations in the uptake of this pathogen from the gut, 2) global spantide II-mediated immune suppression, or 3) nonsubstance P receptor-mediated effects of spantide II on macrophages. The ability of Salmonella to induce substance P receptor expression on cultured macrophages suggested that one mechanism for resistance against this intracellular pathogen might be a direct effect of substance P on this cell population.

Endogenous and exogenous interleukin-12 augment the protective immune response in mice orally challenged with Salmonella dublin.

Following oral challenge with Salmonella dublin, we observed significant increases in interleukin-12 (IL-12) protein expression in the mesenteric lymph nodes. The importance of this endogenous cytokine production in the immune response against S. dublin was demonstrated by in vivo depletion of IL-12 with an anti-IL-12 monoclonal antibody prior to oral S. dublin challenge. Mice pretreated with anti-IL-12 antibody had increased salmonellosis and reduced survival times compared with mice receiving control antibody. Furthermore, administration of exogenous murine recombinant IL-12 dramatically increased survival times of mice challenged orally with S. dublin. Together, these results demonstrate that endogenous and exogenous IL-12 significantly augment the mucosal immune response against the intracellular pathogen S. dublin.