Pharmacological modulation of the antigen-induced expression of the low-affinity IgE receptor (Fc epsilon RII/CD23) on rat alveolar macrophages.

Brown-Norway (BN) rats were sensitized by 3 aerosol exposures to ovalbumin (OA; 10 mg/ml) at days 1, 3 and 14. At day 21, the rats were challenged with the antigen or vehicle by aerosol. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and the expression of Fc epsilon RII/CD23 was assessed by flow cytometry after staining with the BB10 monoclonal antibody. A maximum of 74% of the AM from sensitized and challenged BN rats expressed FC epsilon RII/CD23 24 h after OA exposure, compared to 12% of the cells from rats exposed to vehicle. Sprague-Dawley rats were passively sensitized by intravenous injection of 0.1 or 0.05 ml/kg mouse ascitic fluid containing dinitrophenyl (DNP)-specific monoclonal IgE (2682-1) and after 24 h exposed to an aerosol of 5 mg/ml of DNP-bovine serum albumin for 30 min. In this case also, antigen exposure induced the expression of Fc epsilon RII/CD23 on 75% AM, compared to 17% AM from saline-challenged rats. Such an induction of Fc epsilon RII/CD23 on AM was, however, not observed when the animals were challenged with either histamine, serotonin or acetylcholine by aerosol. The antigen-induced expression of Fc epsilon RII/CD23 on AM was inhibited upon treatment of the rats with ketotifen or beclomethasone. In addition, oral or aerosol administration of respectively BN 50730 or BN 52021 (two structurally unrelated platelet-activating factor antagonists), inhibited the antigen-induced Fc epsilon RII/CD23 expression on AM, indicating the participation of this lipid mediator in this process.     

Properties of N-methyl-N-nitrosourea-resistant, Mex- derivatives of an SV40-immortalized human fibroblast cell line

We have selected two N-methyl-N-nitrosourea (MNU)-resistant derivatives of the SV40-transformed, alkyltransferase-deficient (Mex-) human fibroblast cell line MRC5V1. Both derivatives remain Mex-. They are cross-resistant to methylmethanesulphonate (MMS) and 6-thioguanine (6TG) but not 2,6-diaminopurine. They show increased sensitivity to the bifunctional chloroethylating agent mitozolomide (MTZ). We have transfected MRC5V1 and one of our MNU-resistant lines with the bacterial O6-methylguanine (O6-MeG)-DNA methyltransferase (ada) gene. Transfectants of MRC5V1 are significantly more resistant to MNU but exhibit only a small increase in resistance to MMS and MTZ. Transfectants of the MNU-resistant derivative exhibit only a small additional increase in resistance to MNU, no further increase in resistance to MMS and a large increase in resistance to MTZ. The pattern of resistance to cytotoxic agents of these transfectants suggests that a second mechanism of resistance to MNU, independent of alkyltransferase expression, is operating in our resistant lines. This mechanism apparently enables the cells to tolerate O6-MeG and 6TG, but not chloroethyl adducts in their DNA.     

Apoptosis and cytokine release induced by ionizing or ultraviolet B radiation in primary and immortalized human keratinocytes

We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.     

Ultraviolet-B-induced apoptosis and cytokine release in xeroderma pigmentosum keratinocytes

We have assessed the ability of xeroderma pigmentosum and normal keratinocytes grown out from skin biopsies to undergo apoptosis after irradiation with ultraviolet B. Keratinocytes have been studied from xeroderma pigmentosum complementation groups A (three biopsies), C (three biopsies), D (one biopsy), xeroderma pigmentosum variant (two biopsies), and Cockayne syndrome (one biopsy). The three xeroderma pigmentosum group A and the xeroderma pigmentosum group D samples were at least six times more sensitive than normal cells to ultraviolet B-induced apoptosis. The xeroderma pigmentosum variant samples showed intermediate susceptibility. Xeroderma pigmentosum group C samples proved heterogeneous: one showed high sensitivity to apoptosis, whereas two showed near normal susceptibility. The Cockayne syndrome sample showed the high susceptibility of xeroderma pigmentosum groups A and D only at a higher fluence. These results suggest that the relationships between repair deficiency, apoptosis, and susceptibility to skin cancer are not straightforward. Ultraviolet B-induced skin cancer is also thought to be due in part to ultraviolet B-induced impairment of immune responses. The release of the inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha from cultured xeroderma pigmentosum keratinocytes tended to occur at lower fluences than in normals, but was less extensive, and was more readily inhibited at higher fluences of ultraviolet B.     

Alternative end joining during switch recombination in patients with ataxia-telangiectasia

Ataxia-Telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic diseases with similar cellular phenotypes that are caused by mutations in the recently described ATM (encoding ATM) and NBS1 (encoding p95) genes, respectively. Both disorders are accompanied by immunodeficiency in a majority of patients, but the mechanism involved has as yet not been established. We demonstrate that in cells from A-T patients, the switch (S) recombination junctions are aberrant and characterized by a strong dependence on short sequence homologies and devoid of normally occurring mutations around the breakpoint. A low number of S fragments were generated in cells from NBS patients and showed only limited dependence on sequence identity and mutation frequencies were similar to those observed in normal controls. We propose that ATM and p95 are both involved in the final step(s) in class switch recombination with related, but disparate, functional roles. Thus, the general pathway involved in DNA repair also has a major influence on the immunoglobulin isotype switching process.     

Beta-2-adrenoceptor agonists up-regulate the in vitro Fc epsilon receptor II/CD23 expression on, and release from, the promonocytic cell line U937 and human blood monocytes.

The possible regulatory role of beta 2-adrenoceptor agonists in the modulation of CD23 on the human promonocytic cell line U937 and on human monocytes was investigated. Incubation for 48 h in the presence of interleukin-4 (IL-4; 30 U/ml) induced optimal expression and release of CD23 on both U937 cells and human monocytes. When a beta 2-adrenoceptor agonist, either salbutamol or fenoterol, was added to U937 cells or monocytes both the IL-4-induced CD23 expression and release were markedly up-regulated. This effect was dose-dependent, starting at 10 nM and reaching a maximum at 1 microM final concentration of either salbutamol or fenoterol. The potentiating effect of salbutamol and fenoterol on CD23 expression and release was not observed when a beta-adrenoceptor antagonist, either D,L-propranolol (1 microM) or butoxamine (1 microM), was added to the incubation medium. The alpha-adrenoceptor agonist norepinephrine (1 microM) was ineffective in enhancing the IL-4-induced expression and release of CD23 from U937 cells or human monocytes, demonstrating the specificity of the beta 2-adrenoceptor-mediated effect. In the absence of IL-4, a moderate but significant increase in the CD23 expression on U937 cells and human monocytes by these drugs was observed, as compared to the basal values. These results indicate that, besides their bronchodilator effect, beta 2-adrenoceptor agonists may regulate IgE-dependent processes.     

Inhibition of DNA replication by ionizing radiation is mediated by a trans-acting factor

Gamma irradiation of a human cell line containing an extrachromosomal plasmid results in inhibition of the replication of both genomic and plasmid DNA. This inhibition is observed at doses of radiation which produce an insignificant amount of damage in the plasmid DNA molecules. These results indicate that radiation-induced inhibition of DNA replication is mediated by a trans-acting factor.     

Regulation of switching and production of IgA in human B cells in donors with duplicated alpha1 genes.

IgA is the predominant immunoglobulin class synthesized in humans and can be subdivided into two subclasses, IgA1 and IgA2, each encoded by a separate gene and differentially expressed depending on age and anatomical localization of the producing cells. Duplication of the alpha1 gene is frequently observed in selected populations. As this duplication may serve to enhance IgA-mediated immunity, we determined its effect on switching and production of IgA in human B cells. We developed a nested PCR strategy, involving sequencing the switch (S) alpha2 region, the only human S region not sequenced to date, to assess the proportion of cells switching to IgA1 and IgA2 in vivo. Our results show that there is no difference in the serum and salivary levels of IgA1 and IgA or rate of switching to IgA1 and IgA between normal donors and individuals carrying alpha1 gene duplications, suggesting involvement of a regulatory step in the production of IgA.