Development of polyclonal and monoclonal antibodies for immunoassay and neutralization of human interleukin-4

113

Circulating T cells from four patients with the hyper-IgE syndrome were found to produce significantly lower concentrations of interferon-gamma (IFN-'y) in response to stimulation with phytohemagglutinin (PHA) than did T cells from eight age-matched healthy controls, three patients with atopic dermatitis and one patient with chronic granulomatous disease. A clonal analysis revealed that patients with hyper-IgE syndrome had markedly lower proportions of circulating T cells able to produce IFN--y and tumor necrosis factor-a (TNF-at) in comparison with controls. In contrast, the proportions of peripheral blood T cells able to produce IL4 or IL-2 were not significantly different in patients and controls. All the four patients with hyper-IgE syndrome showed high proportions of circulating CD4' helper T cells able to induce IgE synthesis in allogeneic B cells, as well. Such an activity for IgE synthesis appeared to be positively correlated with IL4 production by T cells and inversely related to the ability of the same T cells to produce IFN-y. Since IFN-y exerts an inhibitory effect on the synthesis of IgE and both IFN-'y and TNF-a play an important role in inflammatory reactions, we suggest that the defective production of IFN-y may be responsible for hyperproduction of IgE and the combined defect of IFN-y and TNF-a may contribute to the undue susceptibility to infections seen in patients with hyper-IgE syndrome.

Section: Methodsmentioning

confidence: 48%

Defective in vitro production of gamma-interferon and tumor necrosis factor-alpha by circulating T cells from patients with the hyper-immunoglobulin E syndrome.

Prete¹,

Tiri²,

Maggi³

et al. 1989

113

“…As has been demonstrated both by in vivo DTH reactions and by in vitro studies, individuals with generalized onchocerciasis exhibit severely impaired and frequently absent cell-mediated immune responses to specific parasite antigen (4)(5)(6)(7)(8)26). Specifically, in studies ofboth African and Latin American onchocerciasis, PBMC from virtually every infected individual were incapable of generating detectable IL-2, IFN-'y, or lymphocyte blastogenic responses when stimulated with parasite antigen (4, 5).…”

Section: Discussionmentioning

confidence: 99%

“…IL-2 was assayed using the CTLL assay as described (25). IL-4 was assayed by ELISA using a polyclonal antibody to IL-4 and the monoclonal antibody 1 1B4 (kindly provided by DNAX Research Institute, Palo Alto, CA) as described (26). In preliminary experiments, the working range of the IL-4 assay was found to be 195-5,000 pg/ml.…”

Section: Introductionmentioning

confidence: 99%

Immunoregulation in onchocerciasis. Functional and phenotypic abnormalities of lymphocyte subsets and changes with therapy.

Freedman¹,

Lujan-Trangay²,

Steel³

et al. 1991

To help define the immunoregulatory defects in patients with onchocerciasis, flow cytometric analysis of circulating lymphocyte subpopulations was performed in parallel with functional assays. No significant differences in CD4/CD8 ratios were seen when microfilariae-positive individuals from Guatemala were compared with Guatemalan controls. However, the infected individuals had significantly increased numbers of circulating CD4+CD45RA+ lymphocytes (mean 383%) when compared with controls (mean 16.0%). Coexpression of the activation marker HLA-DR was significantly increased on CD4+ cells from infected individuals. In contrast, no up-regulation of HLA-DR was seen on CD8+ or CD19+ cells. At 1 year after initiation of treatment with seminnual doses ofthe microfilaricide ivermectin, there were significant increases (P < 0.05) in the percentage of CD4+CD45RA-cells, the percentage of CD4+HLA-DR+ cells, and mitogen-induced lymphokine production (IL-2, IL4). Despite these changes, parasite-specific IL-2 and IL4 production which had been undetectable before treatment did not manifest itself even by the 2-yr follow-up. Defects in the T-cell activation pathway in Onchocerca volwulus-infected individuals may thus exist at several independent points; a state of parasite antigen-specific tolerance appears to remain even after the relative reversal of other generalized immunoregulatory defects. (J. Clin. Invest. 1991. 88:231-238.)

“…Polyvinylchloride "U" bottom microtiter plates were coated with 10 Mg/ml in PBS for 2 h at 37°C with various rat monoclonal anti-IL-3 antibodies. These were produced in vitro in HB102 medium and purified by 66% saturated ammonium sulfate precipitation and AcA44 gel filtration chromatography, essentially as described (40). Plates were washed (three cycles) between all steps with salineTween 20.…”

Section: Methodsmentioning

confidence: 99%

Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3.

Kaushansky¹,

Shoemaker²,

Broudy³

et al. 1992

IL-3 is a glycoprotein cytokine involved in the hematopoietic response to infectious, immunologic, and inflammatory stimuli. In addition, clinical administration of recombinant IL-3 augments recovery in states of natural and treatment-related marrow failure. IL-3 acts by binding to high affinity cell surface receptors present on hematopoietic cells. To determine the site(s) at which IL-3 binds to its receptor, we analyzed a series of interspecies chimera of the growth factor for species-specific receptor binding and biological activity. The results suggest that IL-3 binds to its receptor and triggers a proliferative stimulus through two noncontiguous helical domains located near the amino terminus and the carboxy terminus of the molecule. To corroborate these findings, we have also mapped the binding epitopes of 10 mAb of human or murine IL-3, and have defined four distinct epitopes. Two of these epitopes comprise the amino-terminal receptor binding domain. A third epitope corresponds to the carboxy-terminal receptor interactive domain, and the fourth epitope, apparently not involved in the interaction of IL-3 and its receptor, lies between these sites. And on the basis of sandwich immunoassays using pairs ofthese mAbs, the two receptor interactive regions appear to reside in close juxtaposition in the tertiary structure of the molecule. These results provide a correlation of the structure-function relationships of IL-3 that should prove useful in evaluating the details of IL-3-IL-3 receptor interaction and in the rational design of clinically useful derivatives of this growth factor. (J.