Studies on the ovine mast cell: heterogeneity and involvement in cutaneous inflammation

The distribution of the granule chymase Sheep Mast Cell Proteinase (SMCP) was determined in trachea, bronchus, bronchial lymph node, lung, thymus, spleen, liver, flank skin, abomasum, duodenum, jejunum, ileum, colon and mesenteric lymph node by immunohistochemistry and by ELISA using a polyclonal, affinity purified anti-SMCP antibody. The toluidine blue and SMCP-positive cell counts were closely correlated for all tissues examined (r2= 0.96, P<0.001), with the exception of skin and liver. On the basis of reactivity to the anti-SMCP antibody, two populations of ovine mast cells were identified. SMCP-positive cells (analogous to the gastrointestinal or mucosal mast cell [MMC] subset) were present in all tissues examined whereas SMCP-negative cells were present in skin (the putative ovine connective tissue mast cell [CTMC] subset) and comprised -98% of the ovine dermal mast cell population. The functional heterogeneity of the ovine dermal mast cell population was investigated in cutaneous challenge studies using the secretagogues calcium ionophore A23187 (A23187), substance P (sP) and compound 48/80 (48/80), which are known to activate CTMC subsets in other species. Although only A23187 and sP evoked an immediate weal response (P<0.05; Mann-Whitney U test [MW]), all three agents evoked dermal neutrophil influx (P<0.05; MW) with extensive mast cell degranulation (P<0.05; MW), thus identifying these agents as putative ovine dermal mast cell secretagogues. As SMCP may be released into the dermis following degranulation, its effect in ovine skin in vivo was investigated. SMCP (36pg - 36ng/50pl) evoked a dose-dependent immediate cutaneous response characterized by weal formation (maximal by three hours after injection (P<0.05; MW)) accompanied by dermal neutrophil influx (P<0.05; MW) and concomitant mast cell degranulation (P<0.05; MW). There was no subsequent delayed component to this response (24 to 72 hours). Although heat-inactivation of SMCP (64°C for 10 min; -2% residual activity) abrogated the weal response (P<0.05-P<0.01; MW), there was no effect on dermal neutrophil influx. Recombinant ovine interleukin-3 (rOv.IL-3) was shown to consistently generate a population of rOv.IL-3-dependent bone marrow-derived mast cells (rOv.IL-3 BMMC) in vitro, this cell population being subsequently used to compare functional heterogeneity in vitro to that previously determined in skin in vivo. These generated cells contained the granule-associated mediators arylsulfatase, (^-hexosaminidase and SMCP, the latter finding being consistent with an MMC phenotype. A dose-dependent effect of rOv.IL-3 on cell viability and the maximum percentage of SMCP-positive mast cells obtained was observed (P<0.05-P<0.01; Student's r-test), the latter being increased by transferring the non-adherent cell population to fresh wells or flasks at feeding. When harvested optimally at days 12 to 16 of culture, these rOv.IL-3 BMMC could be activated by sP, 48/80 and A23187 to release arylsulfatase, (3-hexosaminidase and SMCP, indicating that these cells may also possess CTMC characteristics. Thus, r.Ov.IL-3 BMMC may represent a cell population of mixed (MMC and CTMC) phenotype. SMCP failed to evoke similar mediator release in vitro, in contrast to the immediate cutaneous response observed in vivo. One action of SMCP may therefore be to activate vascular endothelium, thereby promoting increased vascular permeability and subsequent dermal neutrophil influx.