Kiri Honma

Determinant analysis of IgE and IgG4 antibodies and T cells specific for bovine αs1-casein from the same patients allergic to cow's milk: Existence of αs1-casein–specific B cells and T cells characteristic in cow's-milk allergy

In an effort to clarify the etiology of milk allergy from the standpoint of allergen-specific immune reactions, we investigated the determinants of IgE, IgG4, and T cells specific for bovine alpha(s)1-casein from the same individual patients by using its synthetic peptides and cyanogen bromide-digested fragments. Alpha(s)1-casein is a major allergen in cows milk, and its unique conformation enabled us to investigate the determinants of antibodies without consideration about missing the reactivities because of conformational changes. Nine patients were selected as subjects from among 129 milk-sensitive infants screened by ELISA to assess the anti-alpha(s)1-casein IgE levels in their sera. By using ELISA for epitope mapping, a C-terminal region of alpha(s)1-casein was identified as a common binding site for IgE from all of these patients, whereas those for anti-alpha(s)1-casein IgG4 were located in multiple regions of alpha(s)1-casein. We determined the specificities of seven alpha(s)1-casein-specific T-cell lines established from peripheral blood mononuclear cells of two of the patients. These T cells have been shown to secrete IL-4. All of the T-cell lines had different specificities to alpha(s)1-casein. However, a common amino acid residue use was found among the determinants of various T-cell lines from each patient. The results suggest that patients allergic to cows milk have characteristic B cells recognizing a limited region of alpha(s)1-casein and secreting alpha(s)1-casein-specific IgE. These B cells may interact particularly with T cells recognizing determinants with a common structure.

Allergenic epitopes of ovalbumin (OVA) in patients with hen's egg allergy : inhibition of basophil histamine release by haptenic ovalbumin peptide

We studied allergenic determinants that induce hypersensitivity to OVA, the major allergen in egg allergy, using immunoblot and histamine release assays. Immunoblot analysis demonstrated a part of the OVA epitope was in the C‐terminal region comprising residues 347‐385 (OVA347‐385). Histamine was released from basophils of a patient with egg allergy upon stimulation with the OVA fragment corresponding to OVA347–385. Furthermore, detailed epitope mapping using overlapping peptides (residues 347‐366, OVA‐A; residues 357‐376, OVA‐B; and residues 367‐385, OVA‐C) in the OVA 347‐385 region was carried out using the histamine release assay. In order for histamine release from basophils to occur, the allergen must possess two or more allergenic determinants located on the protein molecule at distances that would be equivalent to the distances between IgE molecules on the membrane surface. These results suggest that there are at least two epitopes that bind IgE antibodies on each OVA peptide. In addition, one epitope that binds IgE antibodies in two patients appears to reside in the haptenic peptide OVA357‐366 (OVA‐B1). The histamine release from basophils stimulated by OVA‐B was completely inhibited by OVA‐B1 in one of these patients. Similarly, OVA‐B1 inhibited the histamine release produced by OVA‐A in the other by more than 40%. These results suggest that haptenic synthetic peptides could regulate the allergic reaction in the effector phase if common epitope(s) recognized by IgE antibodies in the patients with egg allergy can be found. These are the first studies that provide an antigen‐specific approach to inhibiting histamine release from basophils by a haptenic peptide recognized by IgE antibodies in an allergic disorder.

Specificities of IgE, IgG and IgA Antibodies to Ovalbumin

We studied the binding activities of IgE, IgG and IgA antibodies in patients with allergy to hens egg white against two different ovalbumin (OVA) preparations, which were physically or chemically denatured OVA and enzyme-digested OVA fragments. The binding activities of IgE antibodies to these OVA preparations with those of IgG or IgA antibodies were compared. It was found that the binding activities of IgE antibodies to denatured OVA by treatment with dithiothreitol, urea or hydrochloric acid were similar to those of IgG or IgA antibodies. In contrast, the binding activities of IgE antibodies to heat-denatured OVA or by treatment with sodium hydroxide at pH 11.0 were different from those of IgG or IgA antibodies to these denatured OVA. Furthermore, we found that the binding activities of anti-OVA antibodies in sera from patients with allergy to hens egg white against fragmented OVA were different between IgE antibodies and IgG or IgA antibodies. Thus, it can be concluded that IgE antibodies to OVA in sera from patients with allergy to egg white differ from IgG or IgA antibodies in respect to binding activities against different preparations of denatured or fragmented OVA, probably due to differences in fine specificities of these antibodies against OVA.

Establishment and Characterization of Ovalbumin-Specific T Cell Lines from Patients with Egg Allergy

In order to investigate T cell recognition of allergens in hen egg allergy, we have established 30 ovalbumin (OVA)-specific T cell lines (TCLs) from peripheral blood mononuclear cells of 6 patients with atopic dermatitis, who are positive for IgE antibodies to OVA and clinically allergic to hen egg, and characterized them for their cytokine production pattern. All TCLs we could study were mainly composed of CD4+ T cells. Most TCLs produced significant amounts of interleukin 4 (IL-4) and IL-5 but no or very little interferon gamma on antigen stimulation, suggesting that these TCLs belong to TH2-type T cells. Restriction elements and epitope specificities were further studied on some TCLs. Antibody blocking of the proliferative responses of the TCLs to OVA indicated that HLA-DR are acting as the dominant restriction elements for these TCLs with minor contribution of HLA-DQ. By use of 187 overlapping synthetic peptides covering the whole sequence of OVA, at least 3 different T cell epitopes were identified.

Diminished interferon‐gamma (IFN‐γ) production by bacterial antigen‐specific T cells in atopic patients

In this study, we established and studied cytokine production of T cell lines (TCL) specific to either a purified protein derivative of Mycobacterium tuberculosis (PPD) or Dermatophagoides farinae (Df) from atopic patients and non‐atopic healthy subjects. IFN‐γ was detected in the culture supernatants of all of 36 PPD‐specific TCL established from healthy controls, whereas only 24 of 38 PPD‐specific TCL from patients produced IFN‐γ. Furthermore, the amounts of IFN‐γ produced by PPD‐specific TCL from patients were significantly lower than those from healthy controls. No IL‐4 was detected in any PPD‐specific TCL from either healthy controls or atopic patients. The amounts of IL‐4 production from Df‐specific TCL from atopic patients were much higher than from healthy controls, while few TCL produced IFN‐γ. These results suggest that the skewing to the Th2‐type T cell response in atopic patients is a response not only to allergens, but also to bacterial antigens, compared with non‐atopic subjects. Activation of PPD‐specific TCL from patients with calcium ionophore A23187 plus phorbol myristate acetate resulted in much higher IFN‐γ production than in TCL established from healthy controls, indicating that the low production of IFN‐γ by PPD‐specific T cells from atopic patients is not due to an intrinsic T cell defect but to some regulatory mechanisms.

Comparison of specificity between IgG, IgE and T cells to three casein components: Implication for the role of circulating allergenspecific T cells in food allergy

In order to investigate the role of food antigen‐specific T cells circulating in the blood of patients with food allergy, we compared T cell response to three casein components (αs‐, β‐ and, K‐casein) with specificities of IgG and IgE binding to the casein components in four milk‐allergic patients (P1‐4) with atopic dermatitis. In all patients, the binding activities of IgG antibodies to αs‐casein were most dominant, followed by those to β‐ and to K‐casein. The major component of casein bound by IgE antibodies was αs‐casein in P1 and P3, K‐casein in P2, and αs‐casein as well as K‐casein in P4; the order of casein components bound by IgE antibodies was different from that by IgG antibodies. Proliferative responses of peripheral blood mononuclear cells (PBMC) to casein components were so low that the dominance of casein recognition could not be clearly demonstrated. However, short‐term T cell lines that specifically respond to casein were successfully established from PBMC of the four patients and the proliferative responses of the T cell lines to the three components of casein were in accord with the IgE antibody specificity to casein components but not with that of IgG antibody specificity. When taken together, these results indicate that casein‐specific T cells circulating in the blood are involved in or reflect an allergic reaction against casein.

Establishment and Characterization of αs1-Casein-Specific T Cell Lines from Milk-Allergic Patients

αs1-casein has been found to be one of the major allergens in bovine milk. We isolated 28 αs1-casein-specific T cell lines (TCLs) from 4 milk-allergic patients and characterized their epitope specificites and functions. Stimulation of these αs1-casein-specific TCLs with sequential peptides of αs1-casein revealed the determinants recognized by 5 TCLs. They each reacted to different peptides. In response to αs1-casein, some of them secreted both interleukin 4 (IL-4) and interferon (IFN) -γ, some either IL-4 or IFN-γ only, and the others neither of the two cytokines.