Nerve growth factor prevents apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor

Abstract

We investigated the inhibitory activity of nerve growth factor (NGF) on apoptosis of rat peritoneal mast cells (PMCs) and compared it with that of recombinant stem cell factor (rSCF), which is a mast cell growth factor. When PMCs were incubated up to 72 hours in the presence of control medium, internucleosomal fragmentation of DNA indicating apoptosis was detected by agarose gel electrophoresis and flow cytometry. The aged PMCs showed morphological changes typical for apoptosis, such as chromatin condensation and loss of microvilli of the cell membrane. Addition of NGF or rSCF prevented development of the characteristic DNA fragmentation and decreased the proportion of apoptotic cells with low DNA content values in a dose-dependent manner. Polyclonal antibody to NGF completely abolished the inhibitory activity of NGF but not of rSCF. NGF-induced PMCs were in the G0/G1 phase of the cell cycle, but rSCF transited them from the G0/G1 phase to the S/G2M phase, suggesting that NGF, unlike rSCF, may have no proliferation activity to PMCs. By flow cytometric analysis with antibodies to NGF receptors p75LNGFR and p140trk, we defined that PMCs expressed p140trk but not p75LNGFR. Addition of herbimycin A or K-252a, tyrosine kinase inhibitors, to NGF resulted in blockage of the NGF-induced p140trk phosphorylation and restriction of the inhibitory activity of NGF on apoptosis of PMCs. These results indicated that NGF suppressed apoptosis of rat PMCs through the p140trk tyrosine phosphorylation and possessed no proliferative activity. Thus, NGF may act as a key factor to promote survival of connective tissue-type mast cells.

Nerve growth factor prevents apoptosis of cord blood‐derived human cultured mast cells synergistically with stem cell factor

“…In this study, to check the survival promoting effect of NGF on human mast cells, we investigated the expression of speci®c receptors for NGF on the surface of cord bloodderived human cultured mast cells and a biological activity of NGF for the survival of the cells. The analysis using RT-PCR and¯ow cytometry showed that p140 trk but not p75 LNGFR was expressed on cord blood-derived human cultured mast cells, and immunoprecipitaion and western blot analysis revealed that NGF elicited a rapid phosphorylation of p140 trk on tyrosine residue of the cells, in accordance with the ®ndings on rodent mast cells [20] and recent reports on human mast cells [21,22]. Then we demonstrated that the pretreatment with K252a, a tyrosine kinase inhibitor, inhibited p140 trk kinase activity, suggesting that the effect of NGF was mediated through the p140 trk , as previously described in rat PMC [20].…”

“…Although combination of rhSCF and rIL-6 induced active DNA synthesis, no signi®cant changes on DNA synthesis were observed by the addition of NGF, suggesting that NGF does not have a proliferating activity to human mast cells. We also ascertained that NGF alone could not prevent apoptosis of the cultured human mast cells, unlike rat PMC, of which NGF alone has been reported to support the viability [20]. In contrast, the addition of NGF to the medium containing rhSCF showed a signi®cant inhibitory effect on the apoptosis of the human cultured mast cells.…”

“…Although NGF has been reported to be a survival factor for rat PMC, and to prevent apoptosis of the cells [20,33], human mast cells have been shown not to respond to NGF [21,24]. In this study, to check the survival promoting effect of NGF on human mast cells, we investigated the expression of speci®c receptors for NGF on the surface of cord bloodderived human cultured mast cells and a biological activity of NGF for the survival of the cells.…”

“…Although stem cell factor (SCF) has been identi®ed as a critical human mast cell survival factor, other cytokines which possess survival promotion activity on human mast cells are less known. Recently we have found that NGF is able to suppress apoptosis of rat PMC through p140 trk [20], whereas there has been no report to show the effect of NGF on mast cell survival of other species. Cord blood-derived human cultured mast cells was recently showed to be expressed p140 trk but not p75 LNGFR [21,22].…”

“…To detect the speci®c receptors for NGF on the human cultured mast cells, indirect immuno¯uorescence staining was provided as described previously [20] with slight modi®cation. For p75 LNGFR detection, after washing once, 1´10 6 mast cells were resuspended in 100 mL of ice-cold phosphate-buffered saline (PBS) containing 1% FCS and 0.1% sodium azide with antip75 LNGFR mAb (5 mg/mL) and incubated for 30 min at 4 8C.…”