Memory to the hapten in non-immediate cutaneous allergic reactions to betalactams resides in a lymphocyte subpopulation expressing both CD45RO and CLA markers.

The cutaneous lymphocyte-associated antigen (CLA) is a homing receptor expressed in a subpopulation of memory T lymphocytes that migrates to the skin and participates in different inflammatory processes. The aim of the study was to compare the T cell response to betalactams in both CLA+ and CLA- memory T cell subsets from subjects with non-immediate allergic reactions to these drugs. Three patients with a non-immediate reaction to penicillins were studied during their acute episodes. Peripheral blood mononuclear cells were isolated by Ficoll density gradient and were used for flow cytometry and lymphocyte transformation test assays. CD3+ cells were purified via high affinity negative selection columns. CD45RA+ and CD45RO+ subpopulations were obtained by magnetic sorting and the memory subpopulation was subdivided into CLA+ and CLA- fractions. These were cultured in triplicate together with feeder cells and different concentrations of amoxicillin and benzylpenicillin. In all cases, the proliferative responses to the drugs were confined to the CD45RO+CLA+ subpopulation. The CD45RO+CLA- subset showed no proliferative response to either drug at any concentration. We have shown that the in vitro memory to penicillins in non-immediate cutaneous allergic reactions to these drugs resides in the CD45RO+CD3+ subset expressing CLA, which enables these T cells to migrate to the skin. These findings may have relevance to understanding the involvement of T cells in allergic reactions to penicillins.

Mast cells induce upregulation of P-selectin and intercellular adhesion molecule 1 on carotid endothelial cells in a new in vitro model of mast cell to endothelial cell communication.

It is suggested that mast cells contribute to cell recruitment in inflammation through the upregulation of endothelial adhesion molecules. P-selectin and intercellular adhesion molecule(ICAM)-1 are two key adhesion molecules that have been associated indirectly with mast cell activity. The canine C2 mastocytoma cell line and primary cultures of canine carotid endothelial cells were used to establish a new in vitro model to help study the interaction between mast cells and endothelial cells. Carotid endothelial cells were incubated with mast cell mediators to uncover their effect on endothelial ICAM-1 and P-selectin expression. To assess the relative contributions of tumour necrosis factor (TNF)-alpha and histamine to such effect, an H1 antihistamine and a TNF-alpha blocking antibody were used. Prior to activation by mast cell mediators, P-selectin was expressed only within the cytoplasm, and ICAM-1 was constitutively expressed on the surface of the canine carotid endothelial cells. Both adhesion molecules were enhanced significantly and strongly upon mast cell activation at various time points. Unstored TNF-alpha was fully responsible for ICAM-1 upregulation. P-selectin was up-regulated by both preformed and newly synthesized mast cell mediators, but neither histamine nor TNF-alpha accounted for such an effect. Therefore,a new model is proposed in which the pro-inflammatory effect of mast cells on endothelial cells can be studied in vitro. In this model, it has been demonstrated that only TNF-alpha accounts for the overexpression of ICAM-1 induced by mast cells, and that mast cells up-regulate P-selectin expression through a histamine-independent mechanism.

Expression of the skin-homing receptor in peripheral blood lymphocytes from subjects with nonimmediate cutaneous allergic drug reactions.

BACKGROUND: In nonimmediate cutaneous reactions to drugs, the skin is the organ most frequently involved, and T cells may play a relevant role. T cells related to skin immune responses express the cutaneous lymphocyte-associated antigen (CLA), the skin-homing receptor. METHODS: We studied the expression of the CLA in peripheral blood T cells from nine subjects with exanthematous reactions induced by beta-lactams (4), phenytoin (2), propyphenazone (1), spiramycin plus metronidazol (1), and captopril plus tiazide (1). The cutaneous symptoms appeared at least 6 h after drug intake. CLA expression was evaluated by flow cytometry at the time of the reaction (T1) and 1 month later (T2). HLA-DR activation marker expression was also evaluated at T1. In four patients, it was necessary to readminister the culprit drug to establish a causal relationship, and sequential estimation of the markers was performed. Two control groups were included: healthy controls and subjects exposed to the culprit drugs with good tolerance. Values were compared by nonparametric statistics. RESULTS: The expression of circulating CLA + T cells at T1 was increased compared to healthy controls (median = 20.4 vs 9.4) (P < 0.001), and the patients also expressed increased levels of HLA-DR (median = 3.8) (P < 0.005). Comparison between T1 and T2 (median = 11.2) also showed differences in levels of CLA+ T cells (P < 0.01). The patients re-exposed to the culprit drug showed an increase followed by a decrease of circulating CLA+ T cells (P < 0.05) and CLA+ HLA-DR+ (P < 0.05) paralleling the symptoms. CONCLUSIONS: These data support the immunologic nature of delayed skin reactions to drugs, and suggest that these CLA+ T cells parallel the disease evolution and may participate in the pathophysiologic mechanisms.

Anticonvulsant-induced toxic epidermal necrolysis: monitoring the immunologic response.

BACKGROUND: Toxic epidermal necrolysis is a severe reaction with skin involvement induced by different drugs and other agents. The mechanisms implicated in the induction of the reaction are poorly understood. OBJECTIVE: Our purpose was to study the involvement of T lymphocytes and other immunocompetent cells in the peripheral blood, blister fluid, and affected skin of 3 patients who had a severe reaction after receiving anticonvulsant medication. METHODS: Quantification of T lymphocytes expressing the skin-homing receptor (cutaneous lymphocyte-associated antigen ¿CLA) in peripheral blood, skin, and skin blister fluid and assessment of other adhesion molecules, activation markers, and inflammatory interleukins by flow cytometry, immunohistochemistry, and reverse transcription-PCR. RESULTS: An increase in CD3(+)CLA(+) cells paralleling the severity of the disease was observed in both peripheral blood and skin, tending to normalize as soon as patient's conditions improved. E-selectin was detected in endothelial vessels in parallel with CLA expression on lymphocytes. An overexpression of TNFalpha, IFN-gamma, and IL-2 was also observed in PBMCs. The expression of the different markers changed over the course of the disease. CONCLUSIONS: These data show an increase in activated T cells expressing the skin-homing receptor in both tissue and peripheral blood accompanying clinical symptoms, with a recruitment of macrophages and an overexpression of cytokines. All these results suggest an important role for T cells in the production of toxic epidermal necrolysis.

Rolipram inhibits staphylococcal enterotoxin B-mediated induction of the human skin-homing receptor on T lymphocytes.

The cutaneous lymphocyte-associated antigen defines T lymphocytes with cutaneous tropism under inflammatory conditions. Bacterial infections participate in cutaneous inflammations, such as atopic dermatitis or psoriasis. Bacterial superantigens, such as staphylococcal enterotoxin B, can activate peripheral blood mononuclear cells to induce effector T cells bearing the T cell skin homing receptor cutaneous lymphocyte-associated antigen via enhancement of interleukin-12 production. We have identified and characterized the anti-inflammatory effects of different phosphodiesterase inhibitors on this system. Our data indicate that the selective type 4 phosphodiesterase inhibitor rolipram inhibits the Staphylococcal enterotoxin B-mediated generation of cutaneous lymphocyte-associated antigen positive CD3+ cells from peripheral blood mononuclear cells by reducing interleukin-12 production in a concentration-dependent manner. Conversely, type 3 phosphodiesterase or type 5 phosphodiesterase selective inhibitors were not effective. The rolipram inhibitory effect was on interleukin-12 production, as exogenously added interleukin-12 could revert rolipram suppression. These results suggest that selective type 4 phosphodiesterase inhibition may have beneficial effects on T cell mediated skin inflammatory processes characterized by the presence of bacterial infections, that are thought to exacerbate ongoing skin inflammation.

Circulating CLA+ lymphocytes from children with atopic dermatitis contain an increased percentage of cells bearing staphylococcal-related T-cell receptor variable segments.

BACKGROUND: Atopic dermatitis is an allergic T-cell mediated skin inflammation. Staphylococcus aureus colonization is very common in cutaneous atopic dermatitis lesions. The cutaneous lymphocyte-associated antigen (CLA) is a T cell skin homing receptor that defines T lymphocytes associated with the cutaneous immune response. OBJECTIVE: To study whether CLA+ T cells from atopic dermatitis children present a selective expression for Staphylococcus aureus-related TCR Vbeta segments. METHODS: Peripheral blood T cells were stained with HECA-452 (anti-CLA) and a panel of TCR Vbeta specific monoclonal antibodies and analysed by flow cytometry. RESULTS: Atopic dermatitis patients have a higher percentage of circulating CLA+ CD3+ lymphocytes compared with healthy controls. Patients with active atopic dermatitis during the study expressed a higher percentage of cells positive for the TCR Vbeta2 and Vbeta5.1 segments in the CLA+ but not in the CLA- subset. These TCR Vbetas are recognized by staphylococcal superantigens. Moreover, there was an increased percentage of HLA-DR+ expression by CLA+ Vbeta5.1+ T cells in patients with active atopic dermatitis, but those patients whose eczema was inactive had very similar values to healthy controls regarding TCR Vbeta and HLA-DR phenotype in circulating CLA+ T lymphocytes. CONCLUSION: Our data indicate that circulating skin-homing T cells of patients with active atopic dermatitis contain an increased percentage of cells bearing TCR Vbeta segments related with Staphylococcus aureus. Staphylococcus superantigens may therefore trigger expansion or at least circulation of appropriate CLA+ T cells.

Inhibition of eotaxin-mediated human eosinophil activation and migration by the selective cyclic nucleotide phosphodiesterase type 4 inhibitor rolipram.

1. The effect of the selective type 4 phosphodiesterase (PDE 4) inhibitor rolipram on human eosinophil activation and migration mediated by eotaxin was investigated. 2. Studies were performed with human freshly isolated eosinophils from peripheral blood of healthy donors by a magnetic cell separation (MACS) technique to a purity > 99%. To test the effect of rolipram, eosinophils were stimulated with recombinant human eotaxin and the cell surface activation markers CD11b and L-selectin were analysed by flow cytometry. Furthermore, eotaxin mediated eosinophil migration was measured in a transendothelial chemotaxis assay. 3. Our results indicate that rolipram inhibited eotaxin-induced CD11b up-regulation up to 60.6 +/- 7.6% at the highest tested dose (10 microM), whereas transendothelial chemotaxis was partially inhibited reaching a plateau of approx. 30% at a rolipram concentration of 0.1 microM. 4. We conclude that the selective PDE 4 inhibitor rolipram decreases eotaxin mediated eosinophil activation, an observation that may contribute to elucidate the mechanism by which PDE 4 inhibitors reduce antigen-induced eosinophil infiltration in different animal models of allergic inflammation.

Expression of the cutaneous lymphocyte-associated antigen in circulating T cells in drug-allergic reactions.

BACKGROUND: Mechanisms underlying the production of delayed cutaneous reactions to drugs are poorly characterized. The cutaneous lymphocyte-associated antigen (CLA) is a skin-homing T cell receptor that defines T lymphocytes associated with the cutaneous immune response. We studied the percentage and activation phenotype of circulating CLA+ T cells in drug allergic patients and healthy controls. METHODS: PBMCs were isolated from heparinized venous blood by Ficoll density gradient. Lymphocytes were stained for flow cytometry with anti-CLA, anti-CD3 and anti-HLA-DR mAbs. Five-parameter analysis was performed on an Ortho Cytoron Absolute flow cytometer. RESULTS: We found increased percentages of circulating CLA+ T cells in drug-allergic patients compared to controls. Moreover, CLA+ T cells from drug-allergic individuals expressed a higher percentage of the T cell activation marker HLA-DR. CONCLUSIONS: These results suggest that CLA+ T cells may play a role in the pathology of delayed cutaneous reactions to drugs. Further studies are in progress to elucidate the role of skin-homing T cells in allergic reactions to drugs.

Allergen specificity and endothelial transmigration of T cells in allergic contact dermatitis and atopic dermatitis are associated with the cutaneous lymphocyte antigen.

Recent investigations have indicated a role for antigen-specific T lymphocytes in the local skin immunity. The cutaneous lymphocyte antigen (CLA) is supposed to represent a skin-homing receptor for T cells. Inhibition experiments with specific monoclonal antibody demonstrate that CLA participates in selective transendothelial migration of memory/effector T cells in vitro by interaction with E-selectin on endothelial cell layers after activation with proinflammatory cytokines. In addition, the receptor-ligand pairs VLA-4/VCAM-1 and LFA-1/ICAM-1 are involved in this process. Moreover, only CLA+, CD45RO+ (memory/effector) T cells freshly isolated from peripheral blood of patients with allergic contact dermatitis or atopic dermatitis specifically proliferate in response to the respective allergen. CLA-, CD45RO- T cells from these patients do not respond to the allergens. In contrast, memory T cells from asthmatic individuals and patients with both asthma and atopic dermatitis express the allergen specificity in both T cell subsets. Tetanus toxoid, a systemically acting antigen, also induces a proliferative response in both CLA+ and CLA- memory/effector T cell subsets. These results strongly support the selective role of CLA in homing T cells to the cutaneous tissues and therefore playing a role in the local immunity and inflammatory reactions of the skin.

Antigen focusing by specific monomeric immunoglobulin E bound to CD23 on Epstein-Barr virus-transformed B cells.

Monomeric IgE bound to the low-affinity receptor for IgE (FcERII-CD23) on EBV-transformed human B cells selectively enhances binding of antigen and therefore presentation to specific T-cell clones. To demonstrate the role of monomeric IgE in antigen focusing, we have made use of a system consisting of human T-cell clones specific for Der-P1 (major allergen of the Dermathophagoides pteronyssinus), Der-P1 coupled to NIP (Der-P1-NIP), and the commercially available chimeric (human-murine) monoclonal IgE antibodies with specificity for the hapten NIP. We have found that monomeric IgE binds to CD23 and remains detectable on the surface of the B cells for a period of at least 16 hours at 37 degrees C. Pulsing of these IgE-anti-NIP (1 microgram/ml) treated B cells for 1 hour at 37 degrees C with low amounts (10 ng/ml) of Der-P1-NIP antigen allows the B cells to stimulate Der-P1-specific T cells. Even with IgE concentrations as low as 20 ng/ml, which were not detectable by immunofluorescence, we were able to induce a significant T-cell response. Furthermore, ongoing specific T-cell-B-cell interactions were not inhibited by the presence of high concentrations of nonspecific IgE molecules (incubated with up to 25 micrograms/ml) on the surface of the B cells. Our data confirm the hypothesis that IgE, bound by either CD23 or the high-affinity receptor for IgE, potentiates the immune response. Therefore, IgE may be seen as the fourth general mechanism for antigen capture by (nonspecific) antigen-presenting cells.