Oxazolone-sensitized Mice Research Articles

Ameliorative effect of green odor against UVB-induced immunosuppression of contact hypersensitivity.

Ultraviolet (UV) irradiation to the eye induces photoimmunosuppression. In here, we examined the effect of green odor against immunosuppression of contact hypersensitivity in the eye induced by ultraviolet B (UVB) irradiation. Systemic immunosuppression was induced in ICR mice sensitized with 0.5% oxazolone through the skin by a single exposure to UVB. Consecutive green odor treatment significantly counteracted UVB irradiation-induced immunosuppression of the contact hypersensitivity (CHS) response. The green odor treatment increased dopamine and β-endorphin levels in the brain and the plasma, respectively, and decreased the plasma corticosterone concentration in the oxazolone-sensitized mice after UVB irradiation to the eye, in contrast with that in acetone-treated mice (treatment negative control). Green odor prevented UVB irradiation-induced photoimmunosuppression of the CHS response by regulating the dopamine/β-endorphin/corticosterone pathway.

TLR4 supports the expansion of FasL+CD5+CD1dhi regulatory B cells, which decreases in contact hypersensitivity

Certain B cells termed as “regulatory B cells” (Bregs) can suppress the ongoing immune responses and a splenic CD5+CD1dhi Breg subset identified earlier was shown to exert its regulatory functions through secretion of IL-10. Though FasL expression is an alternative mechanism of immune suppression used by B cells, little is known about the FasL expressing CD5+CD1dhi Bregs. In this study, we isolated splenocytes or splenic CD19+ B cells and compared the efficiency of toll-like receptor(TLR)4 ligand (lipopolysaccharide) with TLR9 ligand (CpG), anti-CD40 and TLR9 ligand (CpG) plus anti-CD40 on the FasL expression of splenic CD5+CD1dhi Bregs by flow cytometry. FasL expression in CD5+CD1dhi B cells was rapidly increased after TLR4 ligation. Intriguingly, anti-CD40 and CpG plus anti-CD40 combinations failed to stimulate FasL expression in CD5+CD1dhi B cells although the IL-10 production was up-regulated in this subset. In addition, LPS and other B10-cell inducers increased the expression of surface molecules like CD86 and CD25, which are correlated to the regulatory functions of B cells. Furthermore, NF-κB and NF-AT inhibitors decreased the TLR4-activated FasL expression in CD5+CD1dhi B cells. Then we sorted splenic CD5+CD1dhi Bregs using flow cytometry and found that TLR4-activated CD5+CD1dhi Bregs suppressed the proliferation of CFSE-labeled CD4+ T cells in vitro, which was partly blocked by anti-FasL antibody. In oxazolone-sensitized mice having contact hypersensitivity, FasL expression in splenic CD5+CD1dhi B cells was decreased compared to the control group after TLR4 ligation. Our findings suggest that the regulatory function of CD5+CD1dhi B cells could be partly mediated by Fas-FasL pathway and this FasL expressing CD5+CD1dhi Bregs might participate in the regulation of inflammatory diseases.

UVA irradiation of the eye modulates the contact hypersensitivity of the skin and intestines by affecting mast cells in mice.

Ultraviolet A (UVA) irradiation before allergic sensitization induces immunosuppression, but the precise mechanism remained unclear. In this study, we examined the influence of UVA irradiation of the eye on contact hypersensitivity (CHS) and the role of mast cells in CHS. We used two types of haptens, fluorescein isothiocyanate (FITC: a Th2 type hapten) and 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone: a Th1 type hapten). A 300 kJ/m(2) dose of UVA irradiation was delivered to the eyes. After UVA irradiation, we sensitized abdominal shaved skin and challenged the ear epidermis and colons of these mice with each hapten. After UVA irradiation, the CHS of the skin and colon were not inhibited in the FITC-sensitized mice. However, in the oxazolone-sensitized mice, only the CHS of the skin was inhibited by UVA irradiation. The inflammation of the colon became more severe after UVA irradiation. In mast cell-deficient (W/Wv) mice sensitized to FITC, the CHS was weaker than that in WT mice. Moreover, the reduction of immunosuppression in ear swelling was seen for one of the two models they used. These results suggest that the mast cells induced by UVA irradiation of the eye have different roles in the epidermis and colon and have different responses to different haptens.

Contact Hypersensitivity to Oxazolone Provokes Vulvar Mechanical Hyperalgesia in Mice

The interplay among pain, allergy and dysregulated inflammation promises to yield significant conceptual advances in immunology and chronic pain. Hapten-mediated contact hypersensitivity reactions are used to model skin allergies in rodents but have not been utilized to study associated changes in pain perception in the affected skin. Here we characterized changes in mechanical hyperalgesia in oxazolone-sensitized female mice challenged with single and repeated labiar skin exposure to oxazolone. Female mice were sensitized with topical oxazolone on their flanks and challenged 1-3 times on the labia. We then measured mechanical sensitivity of the vulvar region with an electronic pressure meter and evaluated expression of inflammatory genes, leukocyte influx and levels of innervation in the labiar tissue. Oxazolone-sensitized mice developed vulvar mechanical hyperalgesia after a single labiar oxazolone challenge. Hyperalgesia lasted up to 24 hours along with local influx of neutrophils, upregulation of inflammatory cytokine gene expression, and increased density of cutaneous labiar nerve fibers. Three daily oxazolone challenges produced vulvar mechanical hyperalgesic responses and increases in nerve density that were detectable up to 5 days post-challenge even after overt inflammation resolved. This persistent vulvar hyperalgesia is resonant with vulvodynia, an understudied chronic pain condition that is remarkably prevalent in 18-60 year-old women. An elevated risk for vulvodynia has been associated with a history of environmental allergies. Our pre-clinical model can be readily adapted to regimens of chronic exposures and long-term assessment of vulvar pain with and without concurrent inflammation to improve our understanding of mechanisms underlying subsets of vulvodynia and to develop new therapeutics for this condition.

Effect of the smell of Seirogan, a wood creosote, on dermal and intestinal mucosal immunity and allergic inflammation

Seirogan, a wood creosote, has been used as an antidiarrhetic drug in Asian countries including Japan for many years. This antidiarrhetic has recently been used as a sugar-coated pill because Seirogan has a strong smell. The strong smell of the uncoated form of Seirogan may modulate the defense systems of animals because the sense of smell is important for the detection of toxic metabolites in foods contaminated with pathogens. This study examined the effect of the sugar-coated and uncoated forms of this antidiarrhetic on the immunological response and inflammatory reactions in mice that had been sensitized with either fluorescein isothiocyanate or oxazolone. The sensitization of mice with either FITC or oxazolone markedly increased the plasma levels of tumor necrosis factor-α and mucosal IgA and elicited severe inflammation in the colon by a mechanism that could be suppressed by exposure of animals to the smell of uncoated Seirogan as effectively as the oral administration of the agent. Dermal inflammation in the FITC- and oxazolone-sensitized mice was also suppressed effectively either by the exposure to the smell or oral administration of the agent. Biochemical and histochemical analyses revealed that the elevated levels of plasma tumor necrosis factor-α and mucosal IgA were significantly decreased by exposure to the smell of uncoated Seirogan as well as by oral administration of the agent. Exposure of mice to the smell of Seirogan but not oral administration of the agent selectively increased plasma levels of adrenocorticotropic hormone and cortisol, particularly in the sensitized animals. These observations suggest that exposing the animals to the smell of Seirogan per se activated the hypothalamo-pituitary-adrenal axis and systemically modulated immunological reactions to suppress the allergic reactions.

Role of Fas/Fas ligand-mediated apoptosis in murine contact hypersensitivity

Apoptosis plays an important role in immune responses, but little is known about its involvement in contact hypersensitivity (CH). In this study, we have investigated the role of Fas/Fas ligand (FasL)-mediated apoptosis in the pathogenesis of CH. Mice were sensitized by one topical application of 100 μl of 3% oxazolone to shaved skin of the abdomen. Six days later, CH was provoked by challenging both sides of sensitized mouse right ear with 15 μl of 1% oxazolone. Using a DNA ladder assay, we found that apoptosis was induced in the skin of oxazolone-sensitized mice 24–96 h after allergen challenge. Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) apoptosis flow cytometric assay showed that early apoptotic CD4 + T cells (annexin V-FITC +PI −), but not late apoptotic CD4 + T cells (annexin V-FITC +PI +), increased in the inflamed skin of mice with CH. Moreover, the expressions of mRNAs for T helper (Th2) cytokine (interleukin (IL)-4), Th1 cytokine (interferon (IFN)-γ) and proapoptotic molecules (Bax, Fas, FasL and IL-1β-converting enzyme (ICE)/caspase-1) were significantly elevated in the oxazolone-sensitized mouse skin 6–72 h after allergen challenge. Dramatic increase in IL-10 mRNA was only observed in the sensitized mouse skin 6 and 12 h after allergen challenge. Furthermore, CH was significantly inhibited with decreased apoptosis and early apoptotic CD4 + T cells in inflamed skin in Fas mutant lpr/ lpr mice compared to wild-type mice, whereas there were no significant differences in IL-4, IFN-γ, IL-10, Bax and ICE mRNAs in the inflamed skin of CH between lpr/ lpr and wild-type mice. Our results thus suggest that Fas/FasL pathway partially contributes to apoptosis in murine CH and that Fas/FasL-mediated apoptosis plays a partial role in the development of CH. The contribution of Fas/FasL-mediated apoptosis to CH appears independent of Th1 and Th2 cytokines.

Development of effector CD8+ T cells in contact hypersensitivity occurs independently of CD4+ T cells.

Contact hypersensitivity (CHS) is a T cell-mediated response to hapten sensitization of the epidermis. Recent results from this laboratory indicated that hapten sensitization induces two populations of hapten-reactive T cells: CD8+ T cells producing IFN-gamma, which mediate the response, and CD4+ T cells producing IL-4 and IL-10, which function to limit the magnitude and the duration of the response. In the current report we first examined the hapten-presenting cell priming each of these T cell populations and then examined the influence of CD4+ T cell priming on the development of the CD8+ effector T cells. Isolation of hapten-presenting Langerhans cells from the lymph nodes of oxazolone-sensitized mice and transfer to naive mice resulted in the induction of both the regulatory CD4+ and the effector CD8+ T populations. Both CD4+ and CD8+ T cells expressing high levels of the activation determinants CD11a and CD44 appeared in the lymph nodes 3 days after hapten sensitization. The CD8+ T cells producing IFN-gamma and mediating CHS responses following transfer to naive mice were restricted to the high CD44-expressing population. In vitro activation of hapten-immune CD8+ T cells resulted in very low amounts (3 U/ml) of IL-2 production, whereas production of IL-2 by immune CD4+ T cells was approximately 70-fold higher (208 U/ml). Despite this discrepancy in IL-2 production and the coincidental priming of CD4+ and CD8+ T cells by hapten-presenting Langerhans cells during hapten sensitization, the numbers of CD8+/high CD44-expressing T cells in the lymph nodes were nearly identical when CD4+ T cells were present or absent during hapten priming. These results indicate that coincidental priming of CD4+ (and CD8+) T cells by LC does not augment CD8+ T cell development in CHS.

Dissection of antigenic and irritative effects of epicutaneously applied haptens in mice. Evidence that not the antigenic component but nonspecific proinflammatory effects of haptens determine the concentration-dependent elicitation of allergic contact dermatitis.

Allergic contact dermatitis differs from most other immune reactions by its strict dose dependence during the elicitation phase. Moreover, almost all known contact allergens can also induce dose-dependent irritative dermatitis and in general only elicit allergic contact dermatitis in sensitized individuals when applied within a narrow dose range. Therefore, we hypothesized that elicitation of contact hypersensitivity (CHS) may require two signals, antigen-specific effector cell activation and a non-antigen-specific proinflammatory signal, both of which are provided by application of a sufficient dose of hapten. To dissociate these putative two signals, oxazolone-sensitized mice were ear challenged with a dose of the specific hapten which was too low to elicit CHS. At the same time, an unrelated hapten was applied in a conventional concentration to the same skin site. Whereas neither treatment alone elicited a significant CHS response, application of both compounds together resulted in a strong CHS response that was indistinguishable from that elicited by the full dose of the specific hapten. Upon coadministration of the irrelevant hapten, allergic contact dermatitis could be elicited even when the dose of the specific hapten was further reduced by a factor of 10(3). In contrast, a dose reduction of the irrelevant hapten by a factor of two resulted in the loss of the CRS response. These data indicate that non-antigen-specific effects of epicutaneously applied haptens significantly contribute to the elicitation of CHS responses and that the capacity of the hapten to evoke this proinflammatory stimulus rather than its antigenicity is responsible for the strict concentration dependence.

Monoclonal antibodies to the integrin α−4 subunit inhibit the murine contact hypersensitivity response

Lymphocyte-endothelial cell recognition is an active multistep process central to the pathophysiology of inflammation. In vitro models of lymphocyte adhesion predict that the beta 1 integrin very late antigen-4 (VLA-4), an activation-dependent adhesion receptor, can mediate the firm sustained attachment required for the extravasation of memory lymphocytes. We have used murine contact hypersensitivity as an in vivo model in which to evaluate the role of alpha-4 integrins in an evolving inflammatory response. We demonstrate that the intravenous administration of 75 micrograms of the anti-alpha-4 specific monoclonal antibodies R1-2 or PS/2 4-6 h prior to challenge significantly inhibits the efferent response of 2,4 dinitrofluorobenzene, or oxazolone-sensitized mice. The disease-modifying effect of anti-alpha 4 treatment was evident as a 50-60% reduction in the ear swelling response. By histological analysis treated animals scored lower for edema, number of epidermal lesions and degree of leukocyte infiltration. Antibody-treated animals have elevated numbers of circulating mononuclear leukocytes present in the same relative ratio as untreated control animals, suggesting that the inhibitory effect was not due to antibody-dependent cellular depletion of effector lymphocytes. These data are consistent with a central role for VLA-4 in the pathophysiologic process of inflammation.

Antigen receptors on murine T lymphocytes in contact sensitivity. II. Presentation and characterization of syngeneic anti-idiotype serum against DNFB-sensitized T cells.

This manuscript describes the preparation and characterization of a syngeneic antiserum raised in BALB/c mice against nylon wool-purified lymph node (LN) T cells from 2,4-dinitrofluorobenzene- (DNFB) sensitized BALB/c mice. This antiserum (anti-T DH-DNP) plus complement blocks the ability of LN or purified T cells from DNFB-sensitized donors to transfer immunity to naive recipients. The inhibitory activity of the serum is due to antibodies; these antibodies have specificity for idiotype (Id) determinants as shown by their ability to bind to purified mouse anti-DNP antibodies but not to rabbit anti-DNP or normal mouse Ig. Inhibition of transfer of immunity by the anti-Id serum is not restricted by either H-2 gene products or Igh-1 allotypes. The antiserum is antigen specific when tested on LN cells from oxazolone-sensitized mice, but has a low level of cross-reactivity against T cells from 2,4,6-trinitrochlorobenzene- (TNCB) sensitized mice. In addition, it was shown that naturally occurring anti-Id antibodies, epsilon-DNP-L-lysine, or DNP-protein (but not NIP-protein) and the anti-Id in syngeneic anti-T DH-DNP serum compete for similar binding sites on DNFB-immune T cells. We interpret these findings to indicate that immunization of mice with T cells from DNFB-sensitized syngeneic mice induces the production of anti-Id antibodies. These anti-Id antibodies are specific for determinants expressed on the T cell receptor or receptor subsite that is specific for the hapten DNP.

Reversal of Pseudomonas aeruginosa-induced disturbance of lymphocyte circulation in oxazolone-sensitized mice by cyclophosphamide

Reversal of Pseudomonas aeruginosa-induced disturbance of lymphocyte circulation in oxazolone-sensitized mice by cyclophosphamide

Disturbance of lymphocyte circulation byPseudomonas aeruginosa infection in oxazolone-sensitized mice

Pseudomonas aeruginosa infection depresses contact sensitivity to oxazolone in mice. To test whether an altered lymphocyte circulation plays a role in this depression51Cr-labeled lymphocytes fromP. aeruginosa-infected and oxazolone-sensitized donors were injected intravenously into infected and sensitized recipients, and the radioactivity uptake of several organs was determine. The controls consisted of normal mice receiving labeled lymphocytes from normal donors. While the radioactivity recovered from the liver, spleen, and mesenteric lymph nodes was similar in the test and the control group, significantly more radioactivity was recovered from the draining lymph nodes of infected and sensitized recipients. The concentration of labeled lymphocytes from sensitized donors in the draining lymph nodes of sensitized recipients was 18% greater than that of the controls but 31% lower than that of infected and sensitized animals receiving cells from infected and sensitized donors.P. aeruginosa infection enhances lymphocyte entrapment within the draining lymph nodes of oxazolone-sensitized mice.

Pseudomonas aeruginosa infection depresses contact sensitivity to oxazolone by enhancing suppressor cell activity

The depression of contact sensitivity to oxazolone in mice infected with Pseudomonas aeruginosa was studied. In oxazolone-sensitized mice, P. aeruginosa infection affects cell proliferation in the lymph nodes draining the site of sensitization. This impaired cell proliferation does not seem to be due to an altered lymphocyte reactivity, since lymph node and spleen cells from infected animals show a normal mitotic responsiveness to both T and B cell mitogens. In addition, the draining lymph nodes and spleens of mice exhibiting a depressed response to oxazolone contain a cell population able actively to suppress the response to the same antigen of syngeneic recipients sensitized immediately before the cell transfer. These suppressor cells require antigenic stimulation and appear to act on the induction phase of contact sensitivity.

Sensitivity to glucocorticoids of lymph node cells stimulated in vivo by oxazolone.

Sensitization of B10.A mice with oxazolone results in delayed-type hypersensitivity (DTH) and a two to three-fold increase in the weight of regional lymph nodes by the third day. Hydrocortisone treatment of the animals 3 days after sensitization suppresses the manifestation of DTH on the tenth day but not on the seventieth day and significantly decreases the weight of lymph nodes both in control and oxazolone-sensitized mice. The regional lymph node cells of oxazolone-stimulated mice were, like the controls, sensitive to dexamethasone in vitro, as judged by viability and thymidine incorporation. Dexamethasone binding capacity of the regional lymph node cells 72 h after oxazolone sensitization increased. However, the dissociation constant for dexamethasone, the binding specificity, and the nuclear transfer of the hormone were not altered. Since high doses of hydrocortisone given 3 days after sensitization were unable to erase permanently the DTH to oxazolone, it seems very likely that cells responsible for the immunological memory survive the high dose of hydrocortisone.

Depression of contact sensitivity by enhancement of suppressor cell activity in Pseudomonas aeruginosa-injected mice.

Heat-killed Pseudomonas aeruginosa depresses contact sensitivity to oxazolone in C56BL/6 mice. The draining lymph nodes and spleens of mice exhibiting an impaired reactivity to oxazolone contain a cell population capable of depressing the response to oxazolone of recipients sensitized immediately before cell transfer. The suppressive activity of these cells appears to be antigen specific, since they do not affect the response to picryl chloride and because they do not arise in P. aeruginosa-injected but not oxazolone-sensitized mice. These suppressor cells occur in the draining lymph nodes and spleen at 3 and 4 days after sensitization, respectively, and have precursors sensitive to cyclophosphamide. It is concluded that P. aeruginosa depresses contact sensitivity to oxazolone by enhancing the suppressor cell activity of the regulatory cells which arise during conventional sensitization.