concanavalin-a and trimellitic-anhydride

The local lymph node assay (LLNA) is used to identify allergens by means of dermal exposure. For hazard identification, besides identification also the distinction between contact and respiratory allergens is of importance. We have previously shown that a modified LLNA can be used to identify respiratory allergens, on the basis of Con A induced IL-4 production. Here we show a good qualitative correlation between mRNA expression and production of IFN-gamma and IL-4. This suggests that distinction between contact and respiratory allergens may also be studied at the mRNA expression level. Secondly, another assay, similar to the modified LLNA but differing in the duration and the number of allergen applications as well as in the ex vivo culture conditions, here denoted as 'longer' assay, has been reported to be able to identify contact allergens, on the basis of (spontaneous) IFN-gamma production. In the present study we have compared these assays. Similar to our previous findings, in the modified LLNA exposure to the respiratory allergen trimellitic anhydride (TMA) resulted in a approximately 10-fold higher Con A induced IL-4 production compared with the contact allergen dinitrochlorobenzene (DNCB), while exposure to both allergens resulted in a similar Con A induced IFN-gamma production. In the 'longer' assay, TMA exposure resulted in Con A induced IL-4 production whereas DNCB exposure did not. Importantly, only a 2-fold higher spontaneous IFN-gamma production was induced by DNCB compared with TMA, the difference being not statistically significant. Thus, although the 'longer' assay indeed showed a somewhat higher IFN-gamma induction by DNCB compared with TMA, the magnitude and robustness of this effect question its applicability. These results favor the modified LLNA since it is shorter, and combines identification of allergens (by cell proliferation) with identification of respiratory allergens (by IL-4 production). Compounds that induce cell proliferation with a low concomitant IL-4 production may thus be identified as contact allergens, although the need to positively identity such allergens remain.

Topics: Allergens; Animals; Concanavalin A; Dinitrochlorobenzene; Enzyme-Linked Immunosorbent Assay; Female; Hypersensitivity; Interferon-gamma; Interleukin-4; Local Lymph Node Assay; Lymph Nodes; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Phthalic Anhydrides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

It has been demonstrated previously that a selective pattern of mitogen-inducible interleukin-4 (IL-4) production becomes apparent in mice after temporal evolution of the immune response to different classes of chemical allergen. Mitogen-stimulated draining lymph node cells (LNC) isolated after primary exposure to both the respiratory allergen trimellitic anhydride (TMA) and oxazolone, a contact allergen, secreted similar amounts of IL-4. Following secondary exposure, however, TMA, but not oxazolone, caused a marked increase in IL-4 production, consistent with the stimulation by TMA of a T-helper type-2 (Th2)-type response. In the present study, cytokine production characteristic of Th1 (interferon-gamma; IFN-gamma) and Th2 (IL-4 and IL-10) cell activation was examined following chronic exposure of mice to allergen over a 13-day period. In accord with previous studies, chronic exposure to TMA, but not to oxazolone, resulted in a substantial potentiation of mitogen-inducible IL-4 secretion. In addition, spontaneous IL-10 production by TMA-activated LNC was significantly higher than by cells prepared from oxazolone-exposed animals. The lower levels of Il-4 and IL-10 elaborated by oxazolone-activated LNC were not attributable to a reduced potential to secrete cytokine per se, as significantly more IFN-gamma was produced compared with TMA-activated LNC. It is proposed that these divergent cytokine production patterns reflect the selective stimulation of Th1- and Th2-type responses by contact and respiratory chemical allergens.

Topics: Allergens; Animals; Cell Division; Concanavalin A; Female; Interferon-gamma; Interleukin-10; Interleukin-4; Interleukins; Kinetics; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Phthalic Anhydrides

It has been demonstrated previously that chemical contact and respiratory allergens differ with respect to the quality of immune responses they will provoke in mice. Trimellitic anhydride (TMA), a human respiratory allergen, induces in mice responses consistent with the preferential activation of Th2-type cells, resulting in the production of IgE anti-hapten antibody and an increase in the serum concentration of IgE. In contrast, oxazolone (OX), a potent contact allergen considered not to cause respiratory hypersensitivity, induces instead Th1-type responses in mice characterized by vigorous IgG2a antibody production and a failure to elicit IgE. In the present study we have extended these investigations and have examined the capacity of these chemicals to stimulate inducible interleukin-4 (IL-4) production by draining lymph node cells (LNC). IL-4 was measured in the supernatants of draining LNC cultured for various periods in the presence or absence of concanavalin A (Con A). Following primary topical exposure to the chemical allergens, Con A-stimulated LNC from OX-treated mice secreted significantly more IL-4 than did LNC from mice exposed to trimellitic anhydride (TMA). A different pattern of IL-4 secretion was observed following culture with Con A of LNC prepared from lymph nodes draining the sites of secondary exposure to these chemicals. In this case significantly higher concentrations of IL-4 were produced by TMA-treated mice. Detectable levels of IL-4 (> 300 pg/ml) were not found following culture of draining LNC from sensitized mice in the absence of Con A or following culture of LNC from naive mice with or without Con A. These data demonstrate that chemical allergens of different types stimulate discrete and changing patterns of inducible IL-4 synthesis consistent with the selective activation of Th-cell subpopulations.

Topics: Allergens; Animals; Cell Division; Cells, Cultured; Concanavalin A; Interleukin-4; Kinetics; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Phthalic Anhydrides