Nerve growth factor induces the expression of certain cytokine genes and bcl-2 in mast cells. Potential role in survival promotion.

Nerve growth factor (NGF) promotes mast cell survival in vitro (Horigome, K., Bullock, E. D., and Johnson, E. M., Jr. (1994) J. Biol. Chem. 269, 2695-2702). NGF survival promotion is cell density-dependent, and conditioned medium experiments have shown that NGF increases the production of an autocrine mast cell survival activity. Cytokines are potential candidates for autocrine survival factors. In rat peritoneal mast cells (RPMC), NGF caused an increase in the messenger RNAs for interleukin (IL)-3, IL-4, IL-10, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor. This induction was NGF dose-dependent, was blocked by NGF-neutralizing antibodies, and was not observed in the non-mast peritoneal cell population. The immunosuppressive agent, cyclosporin A, blocked both cytokine induction and NGF-activated survival promotion but not survival promotion activated by IL-3 or stem cell factor, suggesting that NGF enhanced RPMC survival by increasing cytokine production. We also examine the effects of NGF on the expression levels of some members of the bcl-2 family and the interleukin-1beta-converting enzyme-like cysteine protease families. NGF markedly increased bcl-2 expression but had little or no effect on the other genes studied. The induction of bcl-2 mRNA by NGF was not blocked by cyclosporin A. These data suggest that induced cytokine gene expression but not increased expression of bcl-2 mediates NGF-survival promotion in RPMC.

Antigen-driven differentiation of naive Ig-transgenic B cells in vitro.

We have established a culture system in which naive B cells bearing a transgenic, chicken OVA (cOVA)-specific Ig differentiate to plasma cells in vitro after interaction with cOVA plus cOVA-specific helper T cells. B cell-enriched populations from Ig-transgenic mice, but not from nontransgenic mice, proliferated after presenting nanomolar concentrations of cross-linked cOVA to DO11.10 (cOVA plus IAd-specific) T cells. After 6 to 9 days of culture with Ag and specific T cells, the B cells acquired a plasma cell phenotype and secreted the transgene-derived Ig at high levels. Engagement of B cell surface Ig was not essential for primary B cell differentiation. Differentiating B cells enlarged, clustered, and acquired two plasma cell markers, Syndecan and CD43. B cell CD45 isoform expression changed: the B220 isoform was lost in a T cell-dependent manner, whereas the CD45RB isoform was gained in a T-independent manner. Although unstimulated B cells survived less than 72 h in vitro, those in Ag-stimulated cultures showed reduced early death, a surge of proliferation at 3 to 5 days, and increased death late in the culture. Using a large population of naive B cells of defined antigenic specificity permits us to study a primary immune response to an Ag, rather than to less physiologic polyclonal stimuli. Because all steps of differentiation occurred in vitro, they are easily accessible for study. This coculture system provides an opportunity to observe Ag-specific T cell-B cell collaboration.

Effects of nerve growth factor on rat peritoneal mast cells. Survival promotion and immediate-early gene induction.

Purified rat peritoneal mast cells in vitro die over a period of 2-6 days in conventional serum-containing medium. As mast cells die, they become pyknotic and undergo DNA fragmentation suggestive of an apoptotic process. Treatment of in vitro mast cells with nerve growth factor (NGF) greatly retards and reduces the death of mast cells (EC50 approximately 1 nM), with no effect on mast cell proliferation. Other neurotrophins have no such effect. NGF also induces the immediate early genes c-fos and NGFI-A with a similar dose dependence. In contrast to the secretagogue activity of NGF, neither the survival-promoting effect nor immediate early gene induction requires lysophosphatidylserine. The ability of NGF to promote mast cell survival is cell density-dependent and appears to be primarily because of induction of the synthesis and/or secretion of an autocrine survival factor by stimulated mast cells. These results suggest that the previously observed effects of NGF on mast cell numbers in vivo may in part be because of enhanced survival and that NGF may be an important mediator of mast cell function in normal and pathological states.

Thymic selection of cytotoxic T cells independent of CD8 alpha-Lck association.

The CD8 alpha cytoplasmic domain associates with p56lck, a nonreceptor protein-tyrosine kinase. The biological relevance of CD8 alpha-Lck association in T cell development was tested with transgenic mice generated to express a CD8 alpha molecule with two amino acid substitutions in its cytoplasmic domain, which abolishes the association of CD8 alpha with Lck. The CD8 alpha mutant was analyzed in a CD8-/- background and in the context of the transgenic 2C T cell receptor. The development and function of CD8+ T cells in these mice were apparently normal. Thus, CD8 alpha-Lck association is not necessary for positive selection, negative selection, or CD8-dependent cytotoxic function.

Mediator release from mast cells by nerve growth factor. Neurotrophin specificity and receptor mediation.

Nerve growth factor causes mediator release from rat peritoneal mass cells in the presence of lysophosphatidylserine. We have investigated the neurotrophin and receptor specificity involved in this response. Nerve growth factor produced a dose-dependent release of [14C]serotonin in the presence of lysophosphatidylserine with an EC50 of approximately 1 nM. Incubation with brain-derived neurotrophic factor and neurotrophin-3 did not produce a response. Northern blot analysis with probes for low affinity nerve growth factor receptor (p75), trkA, trkB, and trkC demonstrated a detectable signal for trkA only. Western blots of trkA immunoprecipitates from mast cell culture lysates, probed with anti-phosphotyrosine antibodies, demonstrated expression of functional TrkA protein. To determine whether p75, trkB, or trkC mRNA was present in amounts below the limit of detection for Northern analysis, a sensitive reverse transcriptase polymerase chain reaction protocol was used; again rat peritoneal mast cells demonstrated only trkA. The predominant form of trkA message expressed in rat peritoneal mast cells was smaller than the neuronal form. An 18-nucleotide exon (coding for 6 amino acids in the extracellular domain) in the neuronal message was not found in the predominant mast cell trkA message. PC12 cells, a rat pheochromocytoma cell line, and dissociated rat sympathetic neurons showed both trkA and p75, but not trkB or trkC. Anterior pituitary expressed both trkB and trkC, but not trkA. To confirm the lack of expression of p75 on mast cells, 125I-nerve growth factor was chemically cross-linked to mast cells or PC12 cells and then immunoprecipitated with a monoclonal antibody specific for p75, 192-IgG; no p75 was detected. Thus, mediator release from rat peritoneal mast cells by nerve growth factor was specific and not a general property of neurotrophins, and the response was modulated through the trkA proto-oncogene. To our knowledge, this is the first description of a bone marrow-derived cell type that expresses trkA at both the mRNA and protein levels. These data provide further evidence that p75 is not necessary for nerve growth factor signal transduction.