![Proliferative responses of immune LNC to FQ-photomodified LC-EC. LNC from three mice immunized with FQ-photomodified EC were cultured in triplicate with LC-EC that were photomodified with the same FQ (LC-EC*) or with nontreated LC-EC (LC-EC). FQ photomodification reduced the [ 3 H]TdR incorporation of LC-EC by about 50%. * Indicates p 0.01 compared with the corresponding LNC plus LC-EC group. Error bars represent the SD.](https://www.researchgate.net/profile/Naohiro-Seo/publication/237765932/figure/fig4/AS:667637084332038@1536188493474/Proliferative-responses-of-immune-LNC-to-FQ-photomodified-LC-EC-LNC-from-three-mice_Q320.jpg)
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Fluoroquinolone-Photomodified Cells That Recognize Usage of TCR Vß13 by Immune T Cells Fluoroquinolone Photoallergy: Exclusive
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Fluoroquinolone antibacterial agents are well known to elicit photosensitivity as an adverse effect, and their cross-reactivity has been clinically documented. The photoallergenicity of fluoroquinolones is mainly derived from their photohaptenic moiety, and photomodification of skin epidermal cells with fluoroquinolones is thought to be an initial step for this photoallergy. Here we have explored, both in vivo and in vitro, T cell responses to fluoroquinolone-photomodified cells, focusing on their photoantigenic cross-reactivity. Cells were derivatized with fluoroquinolones under exposure to UV-A, and fluoroquinolone photoadducts were detected in photomodified cells by immunostaining, flow cytometry, and cell ELISA using fluoroquinolone-specific mAb. T cell-mediated hypersensitivity induced and elicited by s.c. injection of fluoroquinolone-photomodified epidermal cells was cross-reactive among six fluoroquinolones. In addition, lymph node cells from mice sensitized with fluoroquinolone-photomodified cells proliferated well in vitro not only to Langerhans cell-enriched epidermal cells photoderivatized with corresponding fluoroquinolone, but also to those photomodified with any of five other fluoroquinolones, supporting their cross-reactivity. In three fluoroquinolones tested, Th1 populations that expanded after in vitro photoantigenic stimulation of immune lymph node cells expressed the same Vβ13 of TCR. The sensitivity could be transferred by the i.v. administration of this Vβ13+ T cell line into naive recipients, in which a high percentage of Vβ13+ cells infiltrated at the challenge site. These findings suggest that these fluoroquinolones carry the same photoantigenic epitope, which is recognized by Vβ13+ T cells, leading to fluoroquinolone photosensitivity and cross-reactivity.
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The T-lymphocyte proliferative response to pigeon cytochrome c was studied in the mouse. H-2a and H-2k strains were responders to this antigen whereas H-2b, H-2d, H-2f, H-2ja, H-2p, H-2q, H-2r, H-2s, and H-2u strains were low or nonresponders. Genetic mapping demonstrated that two major histocompatibility complex (MHC)-linked Ir genes control the response, one in I-A, the other in I-E/I-C. The major antigenic determinant recognized in this response was localized by cross-stimulations with species variants and cyanogen bromide cleavage fragments of cytochrome c. It was found to be a topographic surface determinant composed of an isoleucine for valine substitution at residue 3, a glutamine for lysine substitution at residue 100 and a lysine for glutamic acid substitution at residue 104. Tobacco hornworm moth cytochrome c, which contains a glutamine at residue 100 but a terminal lysine at residue 103 (one amino acid closer to the glutamine), stimulated pigeon cytochrome c immune T cells better than the immunogen. This result demonstrates for the first time a functional T-cell heteroclitic proliferative response in a system under Ir gene control. Immunization with the cyanogen bromide cleavage fragments revealed that only pigeon cytochrome c fragment 81-104 was immunogenic. This fragment primed for a T-cell proliferative response whose specificity was nearly identical to that of the T-cell response primed for by the whole molecule, suggesting that the glutamine at 100 and the lysine at 104 form the immunodominant portion of the antigenic site. Furthermore, mixing experiments using the two cross-reacting antigens, hippopotamus cytochrome c and Pekin duck or chicken cytochrome c fragment (81-104), each of which contains only one of the two immunodominant substitutions, demonstrated that the T lymphocytes responding to the major antigenic determinant comprise a single family of clones that recognize both amino acids as part of the same determinant. Thus, two complementing MHC-linked Ir genes can control the immune response to a single antigenic determinant.
In vivo and in vitro T-cell-activating ability of murine epidermal cells (EC) was investigated in acutely barrier-disrupted skin by extraction of epidermal lipids with acetone or removal of corneocytes by tape stripping. Contact sensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB) and picryl chloride (PC1) and contact photosensitivity (CPS) to tetrachlorosallcylanilide (TCSA) were significantly augmented when challenged or sensitized at sites treated with acetone 24 h before, compared with the intact skin. CS to DNFB was also enhanced by tape stripping, but not by water rubbing, suggesting that physical stress or a toxic effect of acetone was not responsible for the augmentation. Semi-quantification of TCSA-EC photoadducts showed markedly increased permeability of hapten in the epidermis 24 h after acetone treatment. Bioactive IL-1 was more pronounced in barrier-disrupted than in intact skin. Lymph node T cells from PC1-sensitized mice proliferated significantly more in a hapten-specific and co-stimulatory molecule-dependent manner in response to trinitro-phenylated (TNP) EC from acetone-treated skin than to those from untreated skin. Immunofluorescence staining of epidermal sheets and flow cytometric analysis of dispersed EC showed that subpopulations of Langerhans cells (LC) in acetone-rubbed or tape- stripped skin expressed major histocompatibility complex class II CD54 and CD86 molecules at levels higher than the rest of LC and LC from water-treated or untreated epidermis. Therefore, not only increased permeability of hapten through the epidermis but also altered immune functions of EC potentiate T-cell activation in acute barrier disruption. Such augmentation of immune reactivity may be critical to elimination of environmental noxious agents that penetrate easily into the barrier-disrupted epidermis.Keywords: Langerhans cells, tape stripping, acetone rubbing, contact sensitivity
The hapten, trinitrobenzene sulphonic acid, induced weak B7-1 (CD80) and moderate B7-2 (CD86) expression on Langerhans cells and mRNA expression of both molecules in organ-cultured murine skin. The intradermal injection of hapten-treated epidermal cells induced hapten-specific contact sensitivity in synergic mice. Cells of the keratinocyte cell line, Pam 212, or epidermal cells treated with a mixture of anti-Ia/thy1.2/gamma delta antibody plus complement, did not show any sensitizing ability. When hapten-treated epidermal cells were injected into mice after incubation with anti-B7-2 (CD86) or B7-1 (CD80) antibody the resultant contact sensitivity reaction was decreased to less than 50% of the control reaction, a reduction which was similar to that seen with the anti-ICAM-1 and anti-LFA-1 antibody-induced inhibition of contact sensitivity. Anti-B7-1 (CD80) or anti-B7-2 (CD86) antibody also inhibited hapten-specific lymphocyte proliferation or the allogenic mixed lymphocyte and epidermal cell reaction in vitro, although the inhibitory effect of anti-B7-1 antibody was not as significant as that of anti-B7-2 antibody. These results indicate that costimulatory signals induced by a hapten on epidermal Langerhans cells play an important role in the induction of hapten-specific contact sensitivity in mice.
To investigate the immunogenicity of glycopeptides, a peptide fragment from hen egg lysozyme, HEL(81–96)-Y (here named 1) which is immunogenic in H-2k mice and known to bind to the murine major histocompatibility complex (MHC) class II molecule Ek, was synthesized in five different glycosylated forms. The N-terminal serine of HEL(81–96)-Y was derivatized with D-glucose (2), maltotriose (3), and a branched D-glucose pentasaccharide (4). Furthermore, 1 was prepared with a central serine or asparagine derivatized with the branched D-glucose pentasaccharide (5) and GlcNAc (6), respectively. The ability of the five glycopeptides and the non-glycosylated peptide, labeled with 125I, to bind to the two MHC class II molecules, Ak and Ek, was studied using a gel filtration assay. None of them could bind to Ak. Neither 5 nor 6 were able to bind to Ek. Surprisingly 2, 3 and 4 bound better to Ek than did the non-glycosylated peptide 1. The increased binding varied depending on the type of oligosaccharide attached to the N terminus of the peptide. The better binding to Ek of glycopeptide 4 was found to be due to an increased association rate. The binding of 1 as well as 4 was optimal at pH 5.0. Functional studies showed that 4 was able to elicit a heteroclitic proliferative response from T cells of mice immunized with the native non-glycosylated peptide. Circular dichroism studies of 1 and 4 indicated a more unordered structure of 4 and a predominant α-helical conformation of 1, suggesting that the MHC class II molecule may bind to peptides which are in a non-α-helical conformation. These results demonstrate that glycosylation has considerable influence on peptide immunogenicity for T lymphocytes.
The murine T cell proliferative response to the carboxyl terminal cyanogen bromide cleavage fragment 81-104 of pigeon cytochrome c (cyt) has been studied. Two interesting properties of this response have been previously described. First, T cells from B10.A mice primed with pigeon cyt 81-104 show more vigorous proliferation when restimulated with moth cyt 81-103 than when stimulated with pigeon cyt 81-104; that is, the B10.A T cell response to pigeon shows heteroclitic restimulation by moth. Second, T cells primed with the acetimidyl derivative (Am) of pigeon cyt 81-104 did not cross-react with the unmodified cyt fragments, but Am-moth cyt 81-103 still stimulated Am-pigeon cyt 81-104 primed T cells better than the Am-pigeon cyt 81-104 fragment. These results raised the issue of whether the antigenic sites on the fragments responsible for the specificity of T cell priming in vivo differed from the residues that contributed to the heteroclitic response of pigeon (or Am pigeon)-primed T cells to moth cyt c fragments. In this paper, synthetic peptide antigens were tested in order to identify which residues caused the heterocliticity of the moth fragment and which residues were involved in the antigenic differentiation of native and derivatized fragments. The heterocliticity of the T cell response to moth fragment 81-103 was found to be due to the deletion of the penultimate residue (Ala103) from the pigeon fragment. However, the ability to cause heterocliticity was not uniquely a property of this deletion. T cells from animals primed with peptides containing substitutions at positions 100 or 102 were also heteroclitically stimulated by the moth-like antigen. The observation that T cells could not be primed for recognition of the changes in peptide sequence that caused heteroclitic stimulation suggests that T cells do not directly recognize determinants in this region. The antigenically significant site of derivatization for T cell priming was found to be Lys99. Furthermore, substitution of a Gln at position 99 also resulted in elicitation of yet a third set of T cell clones specific for the presence of that residue. That is, the specificity of the primed T cell population was found to be altered by changes at residue-99, but no such alterations in specificity were demonstrable when T cells primed with peptides altered at residue-103, residue-102, or residue-100 were compared. Overall, the results demonstrate that the antigen can be divided into two functionally distinct sites that are in close physical proximity.
The photoreactions of 3,3',4',5-tetrachlorosalicylanilide anion (TCSA-) with two serum proteins were studied. TCSA- and anions of two similar compounds, N-ethyl-3,5-dichlorosalicylamide and salicylanilide, bind noncovalently to human serum albumin (HSA) without irradiation in aqueous pH 7.4 buffered solutions. TCSA- noncovalently bound to HSA yields three types of photoproducts when irradiated with ultraviolet light (lambda greater than 360 nm). A covalently bonded photoadduct between TCSA- and HSA is formed and histidines in HSA are chemically modified. In addition to these two types of photoproducts which involve HSA, two of the four TCSA- photoproducts which form when HSA is absent are also formed when the TCSA-/HSA complex is irradiated. The results presented indicate that not all proteins in the skin are capable of being the carrier protein in photoallergy of TCSA- and that cross reactivity to other halogenated salicylanilides can be explained by further photochemical reactions of TCSA- photoproducts.
We report the results of scarified photopatch testing in 3 patients with photosensitivity to lomefloxacin, a new quinolone antibacterial agent introduced onto the Japanese market in April 1990. The patients developed pruritic eczematous lesions on sun-exposed areas 5 days to 3 weeks after they had started taking lomefloxacin. 2 cases reacted positively to lomefloxacin on scarified photopatch testing down to 0.1% pet., and 1 patient down to 10% pet. One of them also showed positive reactions on conventional photopatch testing. Scarified photopatch testing with 7 other pyridone carbonate derivatives in 2 of the 3 cases, however, did not show any positive reactions. Patch and scarified patch testing were negative in all patients. The clinical course, clinical and histological findings and results of skin tests in these patients suggest that photoallergic mechanisms are involved. This is the 1st report of positive reactions on scarified photopatch testing, which appears to be a useful method of detecting the causative drug in drug-induced photosensitivity.