Study of granule reappearance and histamine synthesis in rat mast cells maintained in short-term cultures

A single cutaneous application of components of poison oak or ivy urushiol oils to mice results in contact sensitivity with properties of delayed-type hypersensitivity. The compounds, 3-heptadecylcatechol (HDC, from poison oak urushiol) and 3-pentadecylcatechol (PDC, from poison ivy urushiol) are completely cross-reactive. Covalent bond formation between the o-quinone intermediate of PDC and nucleophilic functionalities such as those found on proteins is known to occur in a regiospecific manner. Amino nucleophiles preferentially attack the 5-position on the catechol ring while thiol nucleophiles attack the 6-position. The present paper describes the immunological properties of the three possible ring monomethylated analogs of PDC. When mice were treated with a single epicutaneous painting with these analogs, only the 5-methyl compound (5-Me-PDC) was found to be an ineffective sensitizer. The 5-Me-PDC analog, however, was capable of inducing cellular proliferation in draining lymph nodes. Furthermore, epicutaneous pretreatment with 5-Me-PDC suppressed the subsequent induction of contact sensitivity to PDC and HDC in an antigen-specific manner. Equivalent treatment with the 6-Me-PDC analog (a good sensitizing agent) resulted in a less consistent and weaker suppressive effect, while the 4-Me-PDC analog did not display any suppressive activity. The suppressive activity could be demonstrated up to 15 days following primary painting with 5-Me-PDC. Lymph node cells obtained from mice 10 days after a single painting with 5-Me-PDC could transfer the suppressive effect. Under certain circumstances 5-Me-PDC could also sensitize, indicating that the analog retains some sensitizing abilities. Hence, blocking the 5-position of the catechol ring results in a major alteration in the type of immune response elicited. Since 5-Me-PDC is specifically blocked in terms of nucleophilic attack by amino groups, it is suggested that attack by amino nucleophiles is of primary importance in the peripheral metabolic processing of these catechols, which in turn determines the outcome of the immune response.     

Component analysis of the urushiol content of poison ivy and poison oak

The partial separation and quantitation of the components of the urushiol fraction of poison ivy and poison oak are discussed. The urushiol fraction of poison ivy is primarily composed of C₁₅ side-chain catechol, while the urushiol extract of poison oak is principally the C₁₇ homolog. The presence of a C₁₇ homolog in ivy and a C₁₅ homolog in poison oak urushiol is also detected. Each of these catechol derivatives contain a mixture of congeners which are partially separated by the GLC system used. In each case the tri-olefinic component occurs in greatest abundance and the mono-olefinic congener is least abundant; no saturated material was detected. The compounds were analyzed as trimethylsilyl derivatives and a qualitative analysis was accomplished by GC-MS.     

Influence of chemical reactivity of urushiol-type haptens on sensitization and the induction of tolerance

Contact sensitization to components of the urushiol oils of poison oak and poison ivy appears to require covalent bond formation between the o-quinones derived from urushiol catechols and nucleophilic groups on proteins. Previous studies using a murine delayed hypersensitivity model demonstrated that 5-methyl-3-pentadecylcatechol (5-Me-PDC) is an epicutaneous tolerogen to the parent compound and a weak sensitizer to itself. To investigate further the structural requirements for sensitization vs suppression, 5,6-dimethyl-3-pentadecylcatechol (5,6-di-Me-PDC) and 4,5,6-trimethylpentadecylcatechol (4,5,6-tri-Me-PDC) were synthesized. The former compound is blocked at both preferred sites for covalent bond formation and the latter is completely blocked towards conjugate addition reactions. These compounds were tested for sensitizing and suppressive ability. Epicutaneous application of both analogs suppressed subsequent induction of sensitization to 3-pentadecylcatechol (PDC) and 3-heptadecylcatechol (HDC). Lymph node cells from animals treated with 5,6-di-Me-PDC could transfer suppression. The dimethyl analog, 5,6-di-Me-PDC, but not the trimethyl analog also exhibited weak sensitizing capacity. The urushiol analogs 5-pentadecylresorcinol (PDR) and 3-heptadecylveratrole (HDV) which cannot form o-quinones were found to be ineffective sensitizers as well. HDV in addition produced no blastogenesis in draining lymph nodes whereas lymph node cell proliferation induced by 4,5,6-tri-Me-PDC followed the same kinetics as previously observed for HDC. PDR elicited weak proliferation with a different time course. These and previous studies indicate that blocking the C5-position on the catechol ring favors the induction of suppression, although some sensitizing capacity may be retained. Covalent bond formation may not be necessary for the induction of active suppressor cell populations.     

Urushiols of poisonous anacardiaceae

GC-MS has been used to analyze and characterize the mixture of bis-trimethylsilyl derivatives of 3-n-alk(en)ylcatechols (urushiol) obtained from certain poisonous members of the Anacardiaceae. Analyses revealed a variation in composition of urushiol obtained from the same species. Furthermore, urushiols from poison ivy and poison wood, while consisting largely of n-C₁₅-substituted catechols, also contain varying amounts of the homologous n-C₁₇-substituted catechols. Similarly, the 3-alk(en)ylcatechol mixtures from poison oak, while containing mainly n-C₁₇-species, also contain varying amounts of the _n-C₁₅ species. The analysis of a single poison sumac sample revealed that it contained predominantly 3-_n-pentadec(en)ylcatechols.     

Enrichment and function of urushiol (poison ivy)-specific T lymphocytes in lesions of allergic contact dermatitis to urushiol

The frequencies of urushiol (poison ivy)-specific T cells were determined in the lesional skin and peripheral blood of patients with allergic contact dermatitis to urushiol. Lesions of urushiol dermatitis were biopsied and the T cells retrieved. Frequencies of Ag-specific cells were determined by limiting dilution assay. Enrichment of urushiol specific T cells was detected in lesional skin or blister fluid of five of five of these donors as well as seven of seven donors with urushiol patch tests. There was also enrichment relative to tetanus toxoid specific T cells in blood and skin of six of seven donors. The frequency of lesional T cells specific for urushiol was less than 1/100 and generally less than 1/1000. Furthermore, the frequency of urushiol specific T cells was often less than 1/10,000 in the peripheral blood during an acute eruption. Autoreactive T cells that proliferated to autologous irradiated PBMC were also enriched in skin lesions relative to blood in 5/5 urushiol patch tests. These autoreactive T cells were expanded and found to be CD4+. It has been proposed that such autoreactive T cells recognize autologous DR and may have a role in immunoregulation. Urushiol-specific T cells were expanded from limiting dilution wells of two donors and characterized. The majority of the urushiol-specific cells were CD8+, and 13/43 lines were suppressive for PWM-induced IgG synthesis.     

The effect of IgE-mediated mast cell degranulation on the expression of experimental contact sensitivity in the mouse

The effects of mast cell activation/degranulation on the elicitation of contact sensitivity (CS) to oxazolone and dinitrofluorobenzene were investigated. Mice were actively sensitized to oxazolone by epicutaneous painting followed by ear challenge. Passive sensitization to DNFB was induced by intradermal injections of dinitrophenol (DNP)-specific cloned T cells in the ears. Mast cells in the challenged ears were activated in various time periods by inducing a passive cutaneous anaphylaxis reaction where passive sensitization with monoclonal IgE anti-DNP antibodies was followed by iv injection of DNP-BSA. This combination of immediate and delayed-type hypersensitivity reactions resulted in a significant increase of ear swelling without any noticeable effect on cellular infiltration when the contact response was evaluated a short time (3-4 hr) after mast cell activation. The very same results were obtained in naive (unsensitized) mice, indicating that this reaction was nonspecific. However, when the CS reaction was evaluated at its peak, i.e., 24 hr post challenge, mast cell activation that had been induced 0.5-11 hr after ear challenge did not have any significant effect on both swelling and cellular infiltration when the latter was evaluated by a radiometric assay. We conclude that in these systems mast cell activation/degranulation makes little or no contribution to the modulation of T-cell activity.     

SUBCUTANEOUS OR ORAL ADMINISTRATION OF rhIL-11 MODULATES THE CONTACT HYPERSENSITIVITY RESPONSE

Recombinant human interleukin 11 (rhIL-11) is a multifunctional cytokine with immunomodulatory activity on both T cells and macrophages. The effects of rhIL-11 in a murine model of contact hypersensitivity (CHS) response have been studied. The CHS response is a T cell-mediated response directed against chemically modified self-proteins following epidermal exposure to haptens. CHS is generated in two phases. The sensitization phase involves dermal dendritic cell recognition of haptenized proteins and antigen presentation. The effector phase involves T cell recognition and activation. In mice sensitized with oxazolone, CHS was induced by secondary challenge to the right ear and measured by ear swelling 24 h later. rhIL-11 significantly suppressed CHS as measured by ear swelling and tissue myeloperoxidase activity when injected subcutaneously for 5 days from the day of sensitization or when administered only on the day before and the day of challenge, but was not effective when administered prior to or on the day of sensitization. These results indicate that subcutaneously administered rhIL-11 may modulate the effector phase of CHS. Administration of rhIL-11 as an oral gavage prior to sensitization also reduced CHS. However oral administration of rhIL-11 after sensitization had no effect. These results suggest that orally and subcutaneously administered rhIL-11 may act through different mechanisms to affect CHS.     

Subcutaneous or oral administration of rhIL-11 modulated the contact hypersensitivity response

Recombinant human interleukin 11 (rhIL-11) is a multifunctional cytokine with immunomodulatory activity on both T cells and macrophages. The effects of rhIL-11 in a murine model of contact hypersensitivity (CHS) response have been studied. The CHS response is a T cell-mediated response directed against chemically modified self-proteins following epidermal exposure to haptens. CHS is generated in two phases. The sensitization phase involves dermal dendritic cell recognition of haptenized proteins and antigen presentation. The effector phase involves T cell recognition and activation. In mice sensitized with oxazolone, CHS was induced by secondary challenge to the right ear and measured by ear swelling 24 h later. rhIL-11 significantly suppressed CHS as measured by ear swelling and tissue myeloperoxidase activity when injected subcutaneously for 5 days from the day of sensitization or when administered only on the day before and the day of challenge, but was not effective when administered prior to or on the day of sensitization. These results indicate that subcutaneously administered rhIL-11 may modulate the effector phase of CHS. Administration of rhIL-11 as an oral gavage prior to sensitization also reduced CHS. However oral administration of rhIL-11 after sensitization had no effect. These results suggest that orally and subcutaneously administered rhIL-11 may act through different mechanisms to affect CHS.     

Conditioned suppression of contact sensitivity is independent of sympathetic splenic innervation

The present study investigated the role of sympathetic innervation of the spleen in conditioned suppression of a contact hypersensitivity (CHS) reaction. Behavioral conditioning was achieved by pairing saccharin drinking solution (conditioned stimulus, CS) with injection of cyclosporin A (CsA, 20 mg/kg; unconditioned stimulus, UCS). Four days after sensitization of the animals by application of a 5% 2,4-dinitrochlorobenzene (DNCB) to abdominal skin, the animals were challenged by applying a 1% DNCB solution to the ear. The CHS response was monitored by measuring the degree of ear swelling. Saccharin re-presentation reduced ear swelling to a magnitude that approached that achieved by CsA treatment. Histological examination demonstrated that the conditioned reduction of ear swelling was produced by a reduced leukocyte infiltration of the ear. Prior sympathetic denervation of the spleen did not alter the conditioned suppression of the CHS response. These data indicate that behavioral conditioning using CsA produces alterations of CHS that, unlike conditioned prolongation of heart allograft survival, are independent of sympathetically regulated conditioned alterations in the spleen.     

Contact hypersensitivity: a simple model for the characterization of disease-site targeting by liposomes

A murine model of delayed-type hypersensitivity (DTH) is characterized with respect to liposome accumulation at a site of inflammation. Mice were sensitized by painting the abdominal region with a solution of 2,4-dinitrofluorobenzene (DNFB) and inflammation was induced 5 days later by challenging the ear with a dilute solution of DNFB. The inflammatory response was readily monitored by measuring ear thickness (edema) and radiolabeled leukocyte infiltration. Maximum ear swelling and cellular infiltration occurred 24 h after the epicutaneous challenge with the ear returning to normal size after approximately 72 h. We demonstrate that large unilamellar vesicles (LUV) accumulate at the site of inflammation to a level more than 20-fold higher than that measured in the untreated ear. Vesicle delivery to the ear correlated with increased vascular leakage resulting from endothelium remodeling in response to DNFB challenge, and was not a consequence of increased local tissue blood volume. Extravasation occurred only during the first 24 h after ear challenge; after this time the permeability of the endothelium to vesicles returned to normal. We further showed that LUV with a diameter of 120 nm exhibit maximum levels of accumulation, that a polyethylene glycol surface coating does not increase delivery, and that the process can be inhibited by the application of topical corticosteroids at the time of induction. These data and the inflammation model are discussed with respect to developing lipid-based drug delivery vehicles designed to accumulate at inflammatory disease sites.     

A role for mast cells and the vasoactive amine serotonin in T cell-dependent immunity to tumors

The involvement of mast cells in anti-tumor resistance was studied by employing 2 strains of mast cell deficient but otherwise immunocompetent mice on a C57BL/6 (H-2b) background (W/Wv and Sl/Sld) and their respective normal +/+ littermate controls. Sensitization of control mice with irradiated semisyngeneic B16 melanoma cells (H-2b) resulted in protection against subsequent challenge with viable B16 cells, in contrast to sensitization of either W/Wv or Sl/Sld mice. The involvement of serotonin in antitumor resistance was studied by employing 2 serotonin active drugs: reserpine, that depletes mast cells of serotonin; and methysergide, a serotonin antagonist. Sensitization of BDF1 mice with irradiated B16 cells and sensitization of DBA/2 mice (H-2d) with irradiated SL2 cells (H-2d) resulted in protection against subsequent challenge with viable B16 cells and viable SL2 cells, respectively. Treatment with either reserpine or methysergide resulted in a decreased protection. Delayed-type hypersensitivity (DTH) footpad responses to allogeneic L5178Y (H-2d) tumor cells in C57BL/6 mice showed a biphasic reaction pattern, similar to that found in DTH responses to simple reactive haptens, such as picryl chloride. Moreover, the early swelling responses were also dependent on T cells and on mast cells. BDF1 mice carrying a semisyngeneic L5178Y tumor on the chest showed an early swelling response after footpad challenge but no late response, possibly indicating that selective down regulation of the late component of DTH was associated with progressive tumor growth in these animals. The biphasic patterns of DTH to both tumor cells and picryl chloride and the T cell and mast cell dependence of both antitumor resistance and DTH to tumor cells suggest that T cell-dependent activation of mast cells to allow entry of mononuclear leukocytes into sites of tumor growth is similar to the mechanism that occurs in DTH.     

Involvement of the cannabinoid CB2 receptor and its endogenous ligand 2-arachidonoylglycerol in oxazolone-induced contact dermatitis in mice

The possible involvement of 2-arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB2), in contact dermatitis in mouse ear was investigated. We found that the level of 2-AG was markedly elevated in the ear following a challenge with oxazolone in sensitized mice. Of note, the swelling following the challenge was suppressed by either the administration of SR144528, a CB2 receptor antagonist, immediately after sensitization, or the administration of SR144528 upon the challenge. The effect of AM251, a CB1 receptor antagonist, was marginal in either case. It seems apparent, therefore, that the CB2 receptor and its endogenous ligand 2-AG are closely involved in both the sensitization phase and the elicitation phase of oxazolone-induced contact dermatitis. In line with this, we found that Langerhans cells (MHC class II(+)) contain a substantial amount of CB2 receptor mRNA, whereas keratinocytes (MHC class II(-)) do not. We also obtained evidence that the expression of mRNAs for proinflammatory cytokines following a challenge with oxazolone was markedly suppressed by treatment with SR144528. We next examined whether the CB2 receptor and 2-AG participate in chronic contact dermatitis accompanied by the infiltration of tissues by eosinophils. The amount of 2-AG in mouse ear dramatically increased following repeated challenge with oxazolone. Importantly, treatment with SR144528 attenuated both the recruitment of eosinophils and ear swelling in chronic contact dermatitis induced by repeated challenge with oxazolone. These results strongly suggest that the CB2 receptor and 2-AG play important stimulative roles in the sensitization, elicitation, and exacerbation of allergic inflammation.     

Phenotypic and functional characterization of ultraviolet radiation-induced regulatory T cells

Sensitization through UV-exposed skin induces regulatory T cells (Treg). In contrast to the classical CD4+CD25+ Treg that act contact dependent, UV-induced Treg (UV-Treg) suppress via IL-10, indicating a distinct subtype that requires further characterization. Depletion studies revealed that UV-Treg express the glucocorticoid-induced TNF family-related receptor (GITR) and the surface molecule neuropilin-1. The injection of T cells from UV-tolerized mice after depletion of UV-Treg into naive recipients enabled a contact hypersensitivity response, indicating that tolerization also induces T effector cells. Adoptive transfer experiments using IL-10-deficient mice indicated that the IL-10 required for suppression is derived from UV-Treg and not from host-derived cells. Activation of UV-Treg is Ag specific, however, once activated suppression is nonspecific (bystander suppression). Hence, speculations exist about the therapeutic potential of Treg generated in response to Ag that are not necessarily the precise Ag driving the pathogenic process. Thus, we studied the consequences of multiple injections of 2,4-dintrofluorobenzene (DNFB)-specific Treg into ears of naive mice followed by multiple DNFB challenges. DNFB-specific Treg were injected once weekly into the left ears of naive mice and DNFB challenge was performed always 24 h later. After three injections, a challenging dose of DNFB was applied on the right ear. This resulted in pronounced ear swelling, indicating that the subsequent boosting of DNFB-specific Treg had caused sensitization of the naive mice against DNFB. These data demonstrate that UV-Treg express GITR and neuropilin-1 and act via bystander suppression. However, constant boosting of Treg with Ag doses in the challenging range results in final sensitization that might limit their therapeutic potential.     

IFN-gamma and TNF-alpha are involved in urushiol-induced contact hypersensitivity in mice

Contact hypersensitivity (CHS) is a cutaneous T-cell-mediated immunological reaction to applied haptens. Activated antigen-specific T cells release several cytokines and chemokines followed by the recruitment of inflammatory cells and skin damage. CD8+ T cells and CD4+ T cells have been involved in the establishment of previously described CHS. In this study, we investigated the induction of CHS by urushiol in mice. Maximum swelling in mouse ears was elicited 24 h after challenge with urushiol on day 9 of sensitization. IFN-gamma, TNF-alpha and IFN-gamma-inducible protein 10 (IP-10) mRNA were expressed after challenge of the antigen in urushiol-sensitized mice, but not in unsensitized mice. IFN-gamma knockout (KO) mice and TNF-alpha KO mice failed to elicit CHS with urushiol. Contact hypersensitivity and expressions of IFN-gamma, TNF-alpha and IP-10 mRNA were markedly suppressed in CD4+ and CD8+ cell-depleted mice. These results suggest that IFN-gamma, TNF-alpha, and possibly IP-10, play a critical role in CHS induced by urushiol, depending on both CD4+ T cells and CD8+ T cells.     

Algerian ivy dermatitis; a California disease

A hitherto unrecognized cause of contact dermatitis in California is the widely cultivated plant known as Algerian ivy. This plant, which grows in California (and its close relative, English ivy) causes a dermatitis which is similar to although milder than that caused by poison oak. Dermatitis from this cause occurs most frequently when persons who have become sensitive to it by previous contact trim the plant back in the spring after its seasonal spurt of growth. For persons whose occupations require repeated contact with the plant, dermatitis from this cause is an occupational hazard. Dermatitis from this plant is easily diagnosed by means of a simple patch test. In a series of 12 cases the only effective treatment was with corticosteroid agents.     

ALGERIAN IVY DERMATITIS—A California Disease

A hitherto unrecognized cause of contact dermatitis in California is the widely cultivated plant known as Algerian ivy. This plant, which grows in California (and its close relative, English ivy) causes a dermatitis which is similar to although milder than that caused by poison oak. Dermatitis from this cause occurs most frequently when persons who have become sensitive to it by previous contact trim the plant back in the spring after its seasonal spurt of growth. For persons whose occupations require repeated contact with the plant, dermatitis from this cause is an occupational hazard. Dermatitis from this plant is easily diagnosed by means of a simple patch test.In a series of 12 cases the only effective treatment was with corticosteroid agents.     

Inhibition of specific immune responses by feeding protein antigens. IV. Evidence for tolerance and specific active suppression of cell-mediated immune responses to ovalbumin.

We studied the effect of a single intragastric administration of ovalbumin (OVA) on the subsequent development of OVA-specific cell-mediated immune (CMI) responses in BDF1 mice. In animals fed OVA 7 days before subcutaneous sensitization with OVA-CFA, we observed a concomitant dose-dependent decrease in both the humoral and CMI responses specific for OVA. The CMI tolerance was found to be antigen-specific when assayed in vivo by ear swelling or in vitro by an antigen-induced T cell proliferation assay because OVA-fed mice responded normally to sensitization with horse gamma-globulin. It was also shown that either spleen or lymph node cells, but not serum, from OVA-fed donors transferred suppression to normal recipients. The transfer was mediated by antigen-specific suppressor T cells (Ts) that appeared to inhibit the induction phase (afferent limb) of the CMI response, since the Ts were only effective when transferred before or shortly after the onset of sensitization.     

Induced Sensitization of Normal Laboratory Animals to Brucella abortus Endotoxin

A Boivin preparation of Brucella abortus, unlike common enterobacterial endotoxins, failed to depress water intake or increase numbers of hemolysin-producing spleen cells in mice, or to cause delayed inflammatory reactions in rabbit skin. Reactivity to the B. abortus endotoxin was found only in animals which were previously given the endotoxin with, but not necessarily in, complete Freund's adjuvant. Previous treatment with the endotoxin in saline or with only the adjuvant was ineffective. Sensitization appeared within 10 days and waned after 5 weeks. Passive sensitization was obtained with sensitized donor spleen cells but not with serum. Serum antibody titers did not correlate with the appearance and disappearance of sensitization.     

Comparative study on picryl chloride (PCL)-induced contact dermatitis in female IQI/Jic and BALB/c mice.

Ear skin responses to picryl chloride (PCL)-induced contact dermatitis were compared in detail between IQI/Jic mice developed in Japan and BALB/c mice often used for the investigation of contact dermatitis. PCL was applied to the left ear of each mouse 4 (1st), 11 (2nd), 18 (3rd) and 25 days (4th) after sensitization of the abdominal skin with PCL. Time course examinations were carried out on the ear swelling responses, total IgE levels, skin histology and immunohistochemistry for infiltrated cells after the 1st and 4th application. In IQI mice, the peak time of the ear swelling responses tended to shift from 24 h to 9 h with marked elevation of total IgE levels and marked increase of mast cells showing degranulation after the 4th application when CD8(+) cells as well as CD4(+) cells also prominently increased. In BALB/c mice, except for the total IgE levels and the number of mast cells, the degrees of ear swelling responses, histological changes and increase of CD4(+) and CD8(+) cells were much less severe. Female IQI mice are considered to be a useful mouse strain for further investigations on the role of CD4(+) and CD8(+) T cells in the pathogenesis of contact dermatitis.     

Induction of histidine decarboxylase in non-mast cells in the spleen of mice by injection of staphylococcal enterotoxin A

Injection of Staphylococcal enterotoxin A (SEA) into WBB6F1-W/WV mice genetically deficient in mast cells resulted in a 10-fold increase in the histidine decarboxylase [HDC, L-histidine carboxylase, EC 4.1.1.22] activity of their spleen. The nature of the spleen cells responsible for this increased HDC activity was studied. The HDC induction by SEA was abolished on day 1 after X-ray irradiation of the mice at 400 rad and restored by transplantation of bone marrow cells from normal WBB6F1-+/+ littermates into the X-ray irradiated WBB6F1-W/WV mice. Transplantation of cells from other organs of the normal mice, such as the thymus, mesenteric lymph node and spleen, did not restore the HDC increase significantly. Transplantation of cultured mast cells also did not restore the increase. Moreover, the high HDC activity of spleen cells induced by SEA was not affected by their treatment with anti-Thy-1,2 antibody and complement. Depletion of phagocytes from the spleen by treatment with carbonyl iron resulted in decrease in HDC activity. These results suggested that phagocytic cells derived from haemopoietic stem cells of the bone marrow were responsible for the increase in HDC activity induced by SEA.