Whether p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascades are required for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in RAW 264.7 murine macrophages exposed to lipopolysaccharide (LPS) plus recombinant interferon-gamma (rIFN-gamma) was investigated. By use of Western blotting for iNOS detection and ELISA for quantitation of TNF secretion, three selective inhibitors of these pathways were tested (the p38 inhibitors SB202190 and SB203580 and the MEK 1,2/ERK inhibitor PD98059). Dose-related inhibition of iNOS production was demonstrated when inhibitors were added 1 h before, simultaneously with, or 1 h after LPS plus rIFN-gamma stimulation. In contrast, inhibition of TNF secretion was observed only when cells were preincubated with these agents. Thus, both the p38 and ERK pathways are involved in the up-regulation of iNOS and TNF production by murine macrophages, and specific inhibitors of these pathways block macrophage iNOS production even when added 1 h after activation of these cells.

The mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and p38, are activated in response to infectious agents and innate immune stimulators such as CpG DNA, and regulate the subsequent initiation and termination of immune responses. CpG DNA activates p38 and ERK with slightly different kinetics in monocytic cells. The present studies investigated the roles of these two key mitogen-activated protein kinases in regulating the CpG DNA-induced production of pro- and anti-inflammatory cytokines in the macrophage-like cell line RAW264.7. p38 activity was essential for the induction of both IL-10 and IL-12 expression by CpG DNA. In contrast, CpG DNA-mediated ERK activation was shown to suppress IL-12 production, but to be essential for the CpG DNA-induced IL-10 production. Studies using rIL-10 and IL-10 gene-deficient mice demonstrated that the inhibitory effect of ERK on CpG DNA-mediated IL-12 production is indirect, due to the role of ERK in mediating IL-10 production. These results demonstrate that ERK and p38 differentially regulate the production of pro- and anti-inflammatory cytokines in APCs that have been activated by CpG DNA. CpG DNA-induced p38 activity is required for the resulting innate immune activation. In contrast, ERK plays a central negative regulatory role in the CpG DNA-mediated Th1 type response by promoting production of the Th2 type cytokine, IL-10.

The mechanisms by which T lymphocytes acquire the capacity to produce interleukin 4 (IL-4) and other lymphokines during intrathymic and extrathymic development are poorly understood. To gain insight into this process, we determined the capacity of human neonatal and adult T lineage cell populations to produce IL4 after polyclonal activation. IL-2 and interferon-y (IFN-'y) production were studied in parallel, since their production by neonatal T cells is known to be similar or diminished, respectively, compared to adult T cells. Production of IL4 by neonatal CD4' T cells and IFN-y by neonatal CD4' and CD8+T cells was markedly lower compared with analogous adult cell populations, whereas IL-2 production was similar. Transcription of IL4, as determined by nuclear run-on assays, and IL4 mRNA-containing cells, as determined by in situ hybridization, were undetectable in neonatal T cells, whereas both were detectable in adult T cells. IFN-'y transcription and IFN--y mRNAcontaining cells were reduced in neonatal T cells compared with adult T cells. Reduced lymphokine production by neonatal T cells correlated with their lack of a CD45R-(putative memory T cell) population; cells with this surface phenotype comprised 30-40% of the adult CD4' T cells and were highly enriched for IL4 and IFN--y, but not IL-2 production. IL-4, IFN-y, and IL-2 mRNA expression by neonatal CD4+CD8-thymocytes was similar to that found in circulating neonatal CD4+ T cells. Taken together, these findings suggest that the extrathymic generation of memory T cells during postnatal life may result in an increased capacity for IL4 and IFN-y gene expression. In addition, IFN-,y and IL-2 mRNA were significantly more abundant than IL4 mRNA in activated neonatal CD4+CD8-thymocytes and CD4+ T cells, as well as adult CD4' CD45R-T cells. Therefore, the capacity of T lineage cells to express the IL4 gene may be more restricted compared to other lymphokine genes beginning in intrathymic development. This restricted capacity appears to persist during postnatal extrathymic maturation of T cells. (J. Clin. Invest. 1991. 87:194-202.).

Pythiosis occurs in animals and humans who encounter aquatic habitats that harbor Pythium insidiosum. Drug therapy for deeply invasive infections with this organism has been ineffective in humans and animals; patients have been cured only by radical surgical debridement. A 2-year-old boy developed periorbital cellulitis unresponsive to antibiotic and antifungal therapy. The cellulitis extended to the nasopharynx, compromising the airway and necessitating a gastrostomy for feeding. P. insidiosum was isolated from surgical biopsy specimens of the affected tissue. On the basis of in vitro susceptibility studies of the isolate, the patient was treated with a combination of terbinafine and itraconazole. The infection resolved over a period of a few months. The patient remained well 1.5 years after completing a 1-year course of therapy. Cure of deep P. insidiosum infection is feasible with drug therapy.