Emeritus Professor Leonie Ashman

Activating mutations in c-Kit, the receptor for Stem Cell Factor (SCF), have been identified in dysplasias and leukaemias of the mast cell lineage and have been shown to contribute to transformation in model systems. Early myeloid cells also normally express c-Kit and their survival, proliferation and differentiation is promoted by SCE It might therefore be expected that c-Kit mutations could also be involved in some acute and/or chronic myeloid leukaemias. We have found that mutant c-Kit (and normal c-Kit in the presence of SCF) provides a strong differentiation stimulus in normal and immortalised murine early myeloid cells. Since maturation of haemopoietic cells, with the exception of mast cells, results in down-regulation of c-Kit expression, the transforming effects of mutant receptor may be self-limiting in most lineages. This is consistent with the observation that multipotential progenitor cells from some patients with systemic mastocytosis express mutant c-Kit. However, c-Kit mutations have been observed in a few cases of myelodysplastic syndromes or AML without mast cell features. Oncogenesis involves multiple genetic changes and the phenotype of malignant haemopoietic cells expressing mutant c-Kit may be influenced by co-oncogenic events. For example mutations blocking the differentiative effect of mutant c-Kit might result in AML rather than mastocytosis. Thus the extent to which c-Kit mutations contribute to malignancies of early myeloid phenotype remains unknown, and resolution of this issue is complicated by the heterogeneity of this family of diseases.

By using reverse transcription-PCR, in situ hybridization, enzyme-linked immunosorbent assay and immunocytochemistry, we have studied the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in human lung mast cells induced by cross-linkage of high-affinity Fce receptors (FceRI). We have also confirmed the bioactivity of GM-CSF released from lung mast cells by investigating the effect of the supernatant from lung mast cells activated with anti-IgE on the release of eosinophil cationic protein (ECP) from eosinophils. Mast cells were purified using affinity magnetic selection with monoclonal antibody (mAb) YB5.B8 (93-99% pure). Purified mast cells were precultured with IgE for 16 h before challenge with 1 µg/ml anti-IgE with or without stem cell factor (SCF). Eosinophils were purified by immunomagnetic negative selection (> 98% pure). The activation of mast cells via FceRI enhanced the intensity of the GM-CSF signal within 2 h and the cells produced GM-CSF protein 4 h after the activation. In the absence of recombinant human (rh) SCF, anti-IgE induced a median GM-CSF response of 202 (< 15 ~ 681) pg/106 mast cells/24 h, whereas in the presence of rhSCF the median IgE-dependent GM-CSF release was 356 (152 ~ 1216) pg/106 mast cells/24 h. This difference was statistically significant (p = 0.0029, n = 12). In contrast, mast cells produced only a small amount of GM-CSF in the absence of anti-IgE. The mast cell supernatant induced ECP release from eosinophils and the release was significantly inhibited by blocking mAb against GM-CSF. These findings indicate that human mast cells are an important cellular source of GM-CSF and as such may contribute to chronic eosinophil-mediated inflammation.

Primary infection of mice with Salmonella enteritidis 11RX (11RX) confers resistance to challenge with 104 LD50 doses of Ehrlich Ascites tumour (EAT). A lipopolysaccharide-free protein extract of 11RX is capable of eliciting delayed type hypersensitivity (DTH) reactions in these mice and of 'recalling' tumour resistance in long-term 11RX immunised mice, which no longer exhibit any resistance to tumour challenge. In the present study, we have examined the ability of five other strains of Enterobacteriaceae to induce similar effects. Primary i.p. injection of S. chester, S. luton or S. typhimurium G30 into mice resulted in persisting infections and the induction of peritoneal exudate cells (PEC) which were tumouricidal in vitro. DTH reactions could also be elicited in these animals with protein extracts of the homologous or the 11RX strain of salmonella. S. friedenan and E. coli K12, which did not persist in mice, did not elicit tumouricidal PEC and did not sensitize mice for DTH reactions. However, protein extracts from all the five strains could elicit tumouricidal PEC and DTH reactions in long-term 11RX-immunised mice (but not in normal mice). The results imply that a wide range of Enterobacteriaceae may possess antigen(s) which can be involved in tumour resistance, provided that these antigen(s) are presented in such a way that a cellular immune response develops.

B16 melanoma maintained by in vivo passage in syngeneic (C57Bl/6) mice was rejected when transplanted into H-2 incompatible BALB/c mice. However, after passage in tissue culture the tumour grew in BALB/c mice and, in the case of intraperitoneal (i.p.) inoculation, was fatal. Administration of lymphoreticular cells (resident peritoneal cells, PC) bearing C57B1/6 alloantigens at the same time as the cultured tumour prevented or greatly diminished tumour growth in BALB/c mice. This occurred when the PC were given either at the same site as the tumour or at a remote site, implying that tumour rejection involved specific sensitization of the BALB/c mice to C57Bl/6 alloantigens presented on the PC. Conversely, administration of PC from a strain (C3H) with a H-2 haplotype unrelated to either the tumour or the allogeneic recipient mice diminished tumour growth when given at the same site, but had no effect when given at a remote site. This result is consistent with the involvement in sensitization of a soluble factor produced by or in response to the allogeneic PC, which is not antigen-specific, and which operates over a short range. These results indicate that, as for mouse thyroid allograft rejection (Lafferty and Woolnough, 1977), rejection of a B16 melanoma allograft was dependent on the presence of donor stimulator cells of lymphoreticular origin. The implications for the generation of immunity in syngeneic and autochthonous tumour systems are discussed.

Administration of a protein antigen preparation from Salmonella enteritidis 11RX at the site of challenge with Lewis lung carcinoma inhibited tumour development in mice previously immunised with live bacteria, but not control mice. The kinetics of tumour growth indicated that the inhibition of tumour development was due to a reduction of the initial tumour inoculum rather than the development of specific antitumour immunity.

The physical, chemical and immunochemical properties of carcinoembryonic antigen (CEA) purified from hepatic metastases of eight tumours, originating in the colon (6), stomach (1) and lung (1), have been examined. Differences were observed in the overall molecular charge, and also in the carbohydrate composition of the different preparations (both total % carbohydrate, and mole % of the individual sugars). Negligible differences in amino acid composition were found. Gel filtration analysis of these CEA preparations and an additional four partially purified preparations (from pancreatic, hepatic, breast and oesophageal tumour tissues) revealed a single CEA-active peak of similar molecular weight (about 200,000-300,000 daltons) in all preparations. Radioimmunoassay data for the twelve CEA preparations indicated that all preparations contain the same antigenic determinants, as detected by our antiserum, but that there are differences in the expression of these determinants in different preparations.

S. enteritidis 11RX infection inhibits the growth of a number of transplantable tumors in mice. In addition, oral infection of mice with S. enteritidis 11RX inhibits colon carcinogenesis by 1,2 dimethylhydrazine. This study has examined the effect of S. enteritidis 11RX infection on two-stage skin carcinogenesis in mice using 7,12 dimethylbenz(a)anthracene (DMBA) as initiator and croton oil as promotor. No protection was observed in either LACA or (BALB/c x C57B1/6J)F1 mice when live 11RX was repeatedly administered i.v. during promotion. When a protein antigen extract from S. enteritidis 11RX was administered i.v. to previously immunized mice during skin carcinogenesis, significant protection was observed both in terms of the number of mice with papillomas and the number of papillomas per mouse. However, the protection was weak and transient. LACA mice were much more susceptible to skin carcinogenesis by DMBA and croton oil than were (BALB/c x C57B1/6J)F1 mice. A preliminary study indicated that BALB/c, C57B1 and CBA mice were also relatively resistant to skin carcinogenesis.

Here we show that the supernatant from activated lung mast cells induced the release of eosinophil cationic protein (ECP) from eosinophils. Lung mast cells were purified using affinity magnetic selection with monoclonal antibody (mAb) YB5.B8 to achieve a final mast cell purity of 93¿99%. Eosinophils were purified by immunomagnetic negative selection (> 98.0% pure). The supernatant was obtained from lung mast cells activated for 24 h with 1 µg/ml anti-IgE and 50 ng/ml stem cell factor (SCF). Human eosinophils were incubated with various concentrations of the supernatants for 4 h and ECP released was measured by RIA. Using 4 different donors¿ supernatant from mast cells, each donor¿s supernatant caused a dose-dependent release of ECP from eosinophils. The dilutant of 1:2 (v/v) of the supernatant induced 657.5 ± 55.6 ng/106 eosinophils of ECP which is statistically significant (p = 0.008, n = 4) compared with the culture medium alone. Anti-interleukin (IL)-5 neutralizing mAb, 10 µg/ml, and anti-tumor necrosis factor-a (TNFa) neutralizing mAb, 10 µg/ml, significantly inhibited the supernatant-induced ECP release in 79.3 ± 9.4 and 68.2 ± 14.1 % (mean ± SEM, n = 6, p < 0.005), respectively. Anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) neutralizing mAb, 50 µg/ml, caused 68.0 ± 6.1% of inhibition (p = 0.002). The isotype negative control had no measurable inhibitory or stimulatory effect for the stimuli. We confirmed that mast cells produce IL-5, GM-CSF and TNFa in response to IgE-dependent stimulation by using RT-PCR, in situ hybridization, ELISA and immunocytochemistry. A million of lung mast cells generated 41.4 pg (7.0¿¿273.6) (median with range) of TNFa, 252.6 pg (158.7¿¿3,652) of GM-CSF and 735 pg (< 10¿¿2,750) of IL-5 24 h after activation with SCF and anti-IgE. These findings indicate that the human mast cells may contribute to the chronicity of tissue inflammation. © 1997 S. Karger AG, Basel.