Molecular cloning of rat interleukin 4 cDNA and analysis of the cytokine repertoire of subsets of CD4⁺ T cells

Abstract

Rat peripheral CD4+ T cells may be subdivided into two functionally distinct subpopulations (OX-22highCD4+ and OX-22lowCD4+) on the basis of their reactivity with the monoclonal antibody MRCOX-22, which recognizes a restricted epitope on the leukocyte common antigen (LCA, CD45). Previous studies have demonstrated the increased activity of the OX-22highCD4+ subset in assays of cell-mediated reactivity, whereas the reciprocal OX-22lowCD4+ subset provides the majority of help for B cells in secondary antibody responses. Analyses of in vivo function have subsequently shown that the autoreactive activity associated with the OX-22highCD4+ subset can be inhibited through a suppressor activity within the OX-22lowCD4+ subset, indicating a further immunoregulatory role for these cells. Since the differential production of lymphokines such as interferon-gamma (IFN-gamma) and interleukin 4 (IL4) is believed to regulate alternative effector responses to a particular antigen, we have compared the lymphokine mRNA profiles of activated OX-22highCD4+ and OX-22lowCD4+ subsets using nucleic acid probes specific for rat IL 2, IFN-gamma and IL4, the latter of which has been isolated by a polymerase chain reaction cloning technique and its sequence is described. A higher frequency of cells expressed IL 2 mRNA in the OX-22high subset, in accordance with the relative levels of IL2 protein produced. In contrast, more IFN-gamma mRNA was detected in the OX-22lowCD4+ subset 24 h after mitogenic stimulation although these cells have consistently been shown to produce less IFN-gamma protein than the OX-22highCD4+ subset. This apparent paradox was resolved by the finding that the IFN-gamma mRNA levels in the OX-22lowCD4+ subset declined rapidly after 24 h while the levels continued to rise in the OX-22highCD4+ population such that at 48 h the relative levels were reversed. We have also demonstrated a higher level of IL4 mRNA expression within the OX-22lowCD4+ subset, which is undoubtedly involved in the increased B cell helper activity mediated by this subpopulation and may be responsible, in part, for their active suppression of cell-mediated immune responses.

Strong expression of CD134 (OX40), a member of the TNF receptor family, in a T helper 2-type cytokine environment

“…We observed an early increase of CD134 expression in this model in a specific subset of CD4 ϩ T lymphocytes, the CD45RC lo cells [21]. Peripheral rat T cells lose CD45RC expression after activation [27]; moreover, most IL-4-producing T cells in the rat are thought to reside within the CD45RC lo subset [28], and these cells are probably responsible for induction of autoimmunity by HgCl 2 [29]. The mechanism by which HgCl 2 exposure enhances CD134 expression in this specific cell subset is unknown.…”

Allergen-Specific Th1 Cells Counteract Efferent Th2 Cell-Dependent Bronchial Hyperresponsiveness and Eosinophilic Inflammation Partly Via IFN-γ

“…Th2-like cells were generated by adding IL-4 (1:100 supernatant from rat IL-4 cDNA-transfected Chinese hamster ovary cell line, a kind gift from N. Barclay, Medical Research Council Cellular Immunology Unit, Oxford, U.K.) (28), and mouse anti-rat IFN-␥ IgG1 Ab (DB1, 20 ng/ml; Serotec) (26). Every 7 days, cells were harvested, washed, and fresh APCs (normal irradiated Brown Norway splenocytes at a ratio of 2:1 for every cultured lymphocyte) along with the same cytokine and Ab mixture, in combination with IL-2, were added.…”

“…PCR was performed on 5 l of diluted cDNA product in a total volume of 25 l with a final concentration of 1ϫ KCl or NH 4 Cl buffer with 1.5 mM MgCl 2 , 0.2 mM dNTP, 0.2 g each of sense and antisense primers, and 1 U Taq polymerase (Bioline, London, U.K.) in a thermal cycler. The primers were designed according to published sequences (28,(33)(34)(35). The PCR reagents were overlaid with mineral oil and amplification was conducted using a multiwell thermal cycler through 20 -40 cycles of denaturation at 94°C for 30 s, annealing at individual temperature for 30 s, and extension at 72°C for 30 s, followed by final extension at 72°C for 10 min.…”

Cytokine expression by inflammatory cells obtained from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis induced by inoculation with myelin basic protein and adjuvants

“…The primers for IL-4 and -actin were those used by McKnight et al (1991). Except for those indicated with *, the sequences are specific for the rat sequences as reported for -actin (Nudel et al, 1983) (Genbank J00691), IL-2 (McKnight et al, 1989) (Genbank M22899), IL4 (McKnight et al, 1991) (Genbank X16058), IL-10 (Feng et al, 1993) (Genbank X60675) and IFN- (Dijkema et al, 1985) (Genbank X0237, X0236, X0235). The sequences indicated by '*' are specific for mouse sequences and differ from the rat sequences by the number of base pairs shown in parentheses.…”