Tumor bearer T cells suppress Bacillus Calmette-Guérin-potentiated antitumor responses. III. Identification of an auxiliary efferent suppressor-T-cell population

T cells (Ts-eff) induced in BALB/c mice by subcutaneous (sc) growth of syngeneic Meth A tumors can adoptively suppress the effector phase of delayed-type hypersensitivity (DTH) in Bacillus Calmette-Guérin (BCG)-primed and unprimed recipients which have been sensitized with irradiated Meth A cells but they do not inhibit the augmented DTH response in recipients inoculated with cyclophosphamide (CY) 2 days prior to sensitization. By reconstituting CY-treated immunized recipients with selected spleen cell populations, it has been demonstrated that Ts-eff suppress DTH by interacting with a second or auxiliary suppressor cell population present in immune but not normal spleens. These auxiliary suppressor cells (Ts-aux) are Thy+, Lyt 1-2+ and I-J+, phenotypically similar to Ts-eff. Their activity is not influenced by B-cell depletion. Unlike Ts-eff, Ts-aux do not bear receptors specific for Meth A cells. Ts-aux and Ts-eff share similar sensitivity to irradiation and high dose (100 mg/kg) CY but unlike Ts-eff, Ts-aux are cortisone sensitive, nondividing, nonadherent cells which are absent from the thymus. The phenotype and mechanism of action of Ts-aux resemble those of the auxiliary or Ts3 cells defined in models of contact sensitivity, DTH to simple haptens, and in vitro antibody responses.     

Schistosoma mansoni: antigen-presenting cells emigrating from skin exposed to attenuated cercariae activate lymphoid cells and transfer protection in C57B1/6 mice

C57B1/6 mice develop significant levels of protection to a challenge infection after percutaneous exposure to irradiated Schistosoma mansoni cercariae. Although some circumstantial evidence has suggested that antigen-presenting cells (APCs) within the skin play a role in priming anti-schistosomula effector mechanisms, no direct evidence has been presented. In this study, we describe efforts to directly test whether skin-resident APCs exposed to irradiated cercariae are capable of mediating responses consistent with previously proposed mechanisms associated with delayed-type hypersensitivity reactions. We demonstrate that a population of APCs emigrates from the skin after percutaneous vaccination and that these cells are able to induce proliferation of S. mansoni-specific lymphocytes. We describe our experiments conducted to confirm that proliferation is dependent on major histocompatibility complex (MHC) Class-II interactions and cell-to-cell contact between APCs and lymphocytes. Immunohistological staining of emigrating cells revealed a population of large MHC Class-II+ cells with a morphology characteristic of mature dendritic cells. On recovery and adoptive transfer into naive mice, these cells demonstrated the ability to mediate protection to a challenge infection at levels similar to those in percutaneously vaccinated controls. This confirms that cutaneous APCs can initiate anti-schistosomula effector mechanisms in C57B1/6 mice after percutaneous vaccination.     

Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. IV. Analysis of the role of IgE antibodies and mast cells

Mice resistant to challenge infection with Schistosoma mansoni by vaccination with highly irradiated cercariae were examined for the presence of circulating IgE antibodies and peritoneal mast cells sensitized against schistosome antigens. Significant levels of SWAP- or CAP-specific IgE antibodies could not be detected by solid phase radioimmunoassay in the sera of C57BL/6 mice during the first 6 wk after vaccination. Similarly, heatlabile antibodies capable of passively sensitizing normal mast cells for degranulation in response to SWAP could not be identified in the same sera. In contrast, peritoneal mast cells harvested from C57BL/6 mice 2 wk or later after vaccination gave strong degranulation responses when challenged with SWAP or CAP. Thus, vaccination with irradiated cercariae induces an unusual form of immediate-type hypersensitivity in which mast cells become sensitized in the absence of detectable circulating IgE antibodies. Mice deficient in mast cells (W/Wv mutant strain) were observed to develop the same resistance to challenge infection after vaccination with irradiated cercariae as nondeficient littermates. Similarly, vaccinated SJL/J mice were found to mount an extremely weak IgE response as measured by mast cell degranulation yet displayed the same level of resistance to challenge infection as other inbred mice developing potent mast cell responses. These findings argue that IgE antibodies and mast cells are not essential components in the effector mechanism of irradiated vaccine-induced immunity against schistosome infection.     

In situ pulmonary responses of T cell and macrophage subpopulations to a challenge infection in mice vaccinated with irradiated cercariae of Schistosoma mansoni

Cellular events related to the resistance induced by radiation-attenuated cercariae of Schistosoma mansoni were determined immunocytochemically in the lung tissues of mice. Thy-1, CD4, CD8, Mac-1 MOMA-1, MOMA-2, and Ia antigens were identified on cryostat sections by the immunogold-silver staining technique with specific monoclonal antibodies. In mice vaccinated with irradiated cercariae and challenged with normal cercariae, the number of Thy-1+ and CD4+ lymphocytes was increased dramatically relative to the normal numbers both in perivascular tissues and in focal cellular aggregates in the parenchyma of the lungs. A high ratio of CD4+/CD8+ T cells was noted in the aggregates, both in perivascular tissues and in the foci. Macrophages showing positive reactions for Mac-1, MOMA-1, MOMA-2, and Ia also infiltrated the foci. In control mice that were unvaccinated and challenged, foci showing positive reactions for the lymphocyte subpopulations barely were detectable in the lungs by day 14. The numbers of Thy-1+, CD4+, and CD8+ cells and the CD4+/CD8+ ratio in controls were considerably less than those in vaccinated/challenged mice over the period of observation. In conclusion, pulmonary cellular aggregates in vaccinated and challenged mice were composed mainly of Thy-1+ and CD4+ cell populations characteristic of delayed-type hypersensitivity (DTH) reactions. Thus, Thy-1+ and CD4+ cells in the lungs of vaccinated mice may be involved in the elimination of challenge parasites through DTH reactions.     

Pulmonary leukocytic responses are linked to the acquired immunity of mice vaccinated with irradiated cercariae of Schistosoma mansoni

Pulmonary cellular responses in C57BL/6 mice exposed to Schistosoma mansoni have been investigated by sampling cells from the respiratory airways with bronchoalveolar lavage. Mice exposed to cercariae attenuated with 20 krad gamma-radiation developed stronger and more persistent pulmonary leukocytic responses than animals exposed to equal numbers of normal parasites. Although vaccination with irradiated cercariae also stimulated T cell responses of greater magnitude and duration than normal infection, the lymphocytic infiltrate elicited by each regimen did not differ substantially in its composition, 5 wk after exposure. Studies with cercariae attenuated by different treatments established that a link exists between the recruitment of leukocytes to the lungs of vaccinated mice and resistance to reinfection. There was a strong association between pulmonary leukocytic responses and the elimination of challenge infections by vaccinated mice. Animals exposed to irradiated cercariae of S. mansoni were resistant to homologous challenge infection but were not protected against Schistosoma margrebowiei. Homologous challenge of vaccinated mice stimulated anamnestic leukocytic and T lymphocytic responses in the lungs, 2 wk postinfection, but exposure of immunized animals to the heterologous species failed to trigger an expansion in these populations of cells. Our studies indicate that pulmonary leukocytes and T lymphocytes are intimately involved in the mechanism of vaccine-induced resistance to S. mansoni. It remains unclear whether these populations of cells initiate protective inflammatory reactions against challenge parasites in the lungs, or accumulate in response to the activation of the protective mechanism by other means.     

In vivo effects of monoclonal anti-L3T4 antibody on immune responsiveness of mice infected with Schistosoma mansoni. Reduction of irradiated cercariae-induced resistance

Mice can be partially protected against challenge infections of Schistosoma mansoni cercariae by either single or multiple exposure to irradiated cercariae (x-cerc). The participation of L3T4+ lymphocytes on this resistance phenomenon was evaluated by selectively depleting this cell population through in vivo administration of mAb anti-L3T4 (hybridoma GK 1.5) at three different times in relationship to the challenge infections. Treatment with anti-L3T4 before challenge such that depletion was effective during the time of cercarial skin penetration and dermal/s.c. residence significantly reduced the level of resistance induced by x-cerc sensitization. When treatment was delayed until after challenge, depletion of L3T4+ cells coincided with either the lung or post-lung/liver phases of schistosomular migration, and normal levels of x-cerc-induced resistance were induced. In contrast to once-immunized mice, mice hyperimmunized by five exposures to x-cerc and then depleted of L3T4+ cells at the time of challenge (skin phase) still expressed resistance to the challenge. These data suggest that when mice are sensitized only once with x-cerc the challenge infection provides a necessary immunologic boost which requires L3T4+ cells for effective expression of resistance. The requirement for this anamnestic effect by the challenge infection can be circumvented by hyperimmunization. Evaluation of the immune response of one-time sensitized or hyperimmunized mice demonstrated that cellular Ag-specific proliferative responses and mitogen-induced lymphokine production were abrogated after any of the various in vivo regimens of anti-L3T4 antibody. In contrast, immunoblot analysis of humoral responsiveness revealed a correlation between the expression of resistance and the ability of sera from immunized and anti-L3T4 treated mice to recognize a 75-kDa parasite antigenic component.     

Resistance in murine schistosomiasis is contingent on activated IL-2 receptor-bearing L3T4+ lymphocytes, negatively regulated by Lyt-2+ cells, and uninfluenced by the presence of IL-4

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. C57BL/6 mice were depleted in vivo of L3T4+, Lyt-1+, Lyt-2+, IL-2R+ cells, or IL-4 by administration of appropriate mAb. Resistance and various correlative parameters of the immune response were studied in normal, depleted, and congenitally athymic mice. Depletion of T lymphocytes by anti-L3T4 or anti-IL-2R mAb reduced the development and expression of resistance, IgG2a and IgE antibody formation, and delayed type hypersensitivity reactivity against schistosome Ag. Depletion with anti-IL-4 antibody led to profound suppression of IgE-eosinophil-mediated antibody-dependent cell-mediated cytotoxicity and passive cutaneous anaphylaxis responses against the parasite and no effect on IgG2a antibody, Ag-mediated blast transformation, or resistance. Depletion of Lyt-2+ cells produced augmented development and expression of resistance and an increase in the immunological parameters of anti-schistosome reactivity. These studies suggest that protective immunity to S. mansoni in mice, induced by irradiated cercariae, is dependent on L3T4+, IL-2R+ lymphocytes and negatively regulated by Lyt-2+ cells. IL-4 does not appear to be essential for the development of resistance but is essential for the IgE response to the parasite.     

In vivo T cell depletion regulates resistance and morbidity in murine schistosomiasis

These studies assessed the roles of subpopulations of T lymphocytes in inducing and modulating resistance to schistosomiasis and thereby influencing subsequent morbidity. C57BL/6 mice were depleted in vivo of Lyt-1+, Lyt-2+, and L3T4+ cells by the daily administration of monoclonal antibodies. The development of protective immunity, induced by exposure to irradiated Schistosoma mansoni cercariae as expressed in depleted animals, was compared to that demonstrated in undepleted, normal, and congenitally athymic C57BL/6 mice. The development of morbidity was determined by spleen weight, portal pressure and reticuloendothelial system activity. The results indicated that depletion of specific subpopulations of T lymphocytes minimally affected the primary development of parasites; however, depletion strongly influenced the development of resistance to the parasite and subsequent morbidity due to infection. Depletion of T lymphocytes by anti-Lyt-1+ or anti-L3T4+ antibody decreased the development of resistance, antibody and delayed-type hypersensitivity directed against schistosome antigens. Morbidity due to disease was increased. Depletion of Lyt-2+ cells produced opposite changes with augmented resistance and reduced morbidity. Congenitally athymic mice developed minimal resistance and morbidity. Moreover, resistance was inversely related to the morbidity shown by a given animal. These studies indicate that the development of protective immunity to S. mansoni cercariae is regulated by discrete subpopulations of T lymphocytes. The feasibility of decreasing morbidity by increasing specific immunologically mediated resistance is suggested.     

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.     

Oral administration of the contact sensitizer trinitrochlorobenzene: initial sensitization and subsequent appearance of a suppressor population

Our earlier studies have demonstrated that intragastric administration of the hapten trinitrochlorobenzene (TNCB) 2 to 3 weeks prior to attempting sensitization with epidermally applied hapten can abrogate development of systemic contact sensitivity (CS). In this paper, we have examined whether onset of tolerance following intragastric administration of the hapten is preceded by development of hapten-specific CS. Indeed, CS was found to be present 5 days after feeding TNCB and in most experiments the response decreased significantly by Days 10 to 12. The kinetics of development of CS by the oral and epidermal routes were strikingly similar except that the magnitude of reactivity (up to 5 days) in orally sensitized mice was somewhat less than that of epidermally sensitized mice. With the exception of Peyer's patches (PP), effector cells of CS were recovered from such gut-associated lymphoid tissues as mesenteric lymph nodes (MLN), lamina propria, and lymphocytes that are present in the intraepithelial compartment of the intestinal wall. These cells as well as spleen cells of TNCB-fed mice were able to adoptively transfer CS to naive mice. The capacity of MLN and spleen cells of TNCB-fed mice to confer CS adoptively was abrogated after treating cells with anti-Thy 1.2 and anti-Lyt 1.1 antibodies plus complement thereby identifying them as T lymphocytes. Although CS decreased by 10-12 days after feeding TNCB, the decline was reversed by pretreating mice with cyclophosphamide (CY) 2 days before giving the hapten. Whereas spleen cells from animals fed hapten 5 days earlier transferred CS readily, those from mice fed hapten 12 days earlier did not. However, when 12-day spleen cells were depleted of Lyt 2+ cells their ability to adoptively transfer CS was restored. These observations indicate that feeding TNCB to mice initially produces CS, mediated by Thy 1.2+, Lyt 1.1+ lymphocytes. CS is subsequently down-regulated by activation of Lyt 2+ suppressor cells, precursors of which are sensitive to CY.     

Suppression of delayed-type hypersensitivity to oxazolone in whole-body-irradiated mice and protection by WR-2721

The effect of whole-body irradiation on cellular immunity, as measured in vivo by delayed-type hypersensitivity (DTH) to oxazolone (4- ethoxymethylene -2-phenyl- oxazol -5-one), was determined in CD2F1 mice. DTH, determined by changes in ear swelling after challenge with oxazolone, was significantly depressed in irradiated mice (500-900 rad of 60Co) in a dose-dependent fashion when animals were irradiated after sensitization and before challenge with oxazolone. Administration of WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] 30 min before irradiation (2 days after sensitization) resulted in protection against suppression of DTH, which was dependent on drug and radiation dose. An effective dose of WR-2721 (200 mg/kg body wt) provided an approximate dose-modifying factor of 1.3. The data suggest that WR-2721 interacts with cells involved in that DTH response (lymphocytes and/or macrophages) and that WR-2721 may be useful in protecting against radiation-induced decrements in cell-mediated immunity.     

Adoptive transfer of protective immunity in experimental schistosomiasis in the mouse

Passive transfer of immune serum alone did not confer protection to recipient mice irrespective of the routes of serum transfer or cercarial challenge of Schistosoma mansoni. Mice that received both sensitized cells and immune serum were protected against challenge by subcutaneous injection of cercariae but not by percutaneous exposure. The immune serum could be transferred as late as 8 days after subcutaneous challenge, suggesting that the protection was afforded in part by a late parasite killing mechanism which functions after the schistosomula have migrated through the lungs.     

Isolation and characterization of a protective antigen for adjuvant-free immunization against Schistosoma mansoni

A single, 68,000 m.w. glycoprotein antigen from adult Schistosoma mansoni was purified by immunoaffinity chromatography with the use of a newly developed, protective, anti-schistosome murine monoclonal antibody. Immunization with two doses of 0.5 microgram or 1 microgram of purified antigen, without adjuvants, afforded a mean 28% reduction in parasite recovery in CF1 mice, and 2-% reduction in parasite BALB/c mice. On immunoblotting, the 68,000 m.w. antigen was common to S. mansoni adults and schistosomula, whereas parasite eggs contained only cross-reacting low m.w. antigens of 19,100 and 16,000. Immunization resulted in the development of anti-antigen antibody and enhanced immediate cutaneous hypersensitivity to the 31-3B6 antigen. By contrast, delayed-type hypersensitivity and sensitization to circumoval granuloma formation were not observed in immunized mice. It was concluded that the 68,000 m.w. 31-3B6 antigen represents a candidate vaccine for adjuvant-free immunization against S. mansoni.     

A comparative study of the death of schistosomula of Schistosoma haematobium and Schistosoma mansoni in the skin of mice and hamsters.

This study shows that some cercariae of S. haematobium and S. mansoni die during penetration of mouse or hamster skin. Approximately 30-38% of cercariae of both species die in mouse skin and 14-16% die in hamster skin. The greater number of cercariae which die in the skin of mice seems to account for the higher yield of adult worms recovered in hamsters. Adult worm recoveries from animals infected with S. haematobium were, however, only about half the worm recoveries from hosts infected with S. mansoni.     

The regulation of resistance to Schistosoma mansoni by auto-anti-idiotypic immunity. III. An analysis of effects on epitopic recognition, idiotypic expression, and anti-idiotypic reactivity at the clonal level.

Auto-anti-idiotypic mechanisms can regulate the protective immune response against Schistosoma mansoni. Anti-idiotypic responses were stimulated by immunization of mice either with nonspecifically induced lymphoblasts, produced with Con A, or with Ag-induced lymphoblasts bearing specific idiotypic receptors. The effect of the induced anti-idiotypic response upon clonotypic cellular reactivity was assessed in vitro through the suppression of antigen-mediated blast transformation by cloned T cells and in vivo by suppression of resistance to S. mansoni and delayed-type hypersensitivity responses against specific Ag. Differential regulation of humoral immune responses was studied at the levels of specific epitopic recognition, the expression of specific Id, and the production of anti-idiotypic responses directed against mAb bearing specific Id. Anti-idiotypic sensitization resulted in variable (10 to 90%) suppression of the immune response to discrete antigenic epitopes, the expression of specific idiotypic phenotypes, and anti-idiotypic, antiparatopic responses against T cell clonotypes and antibody idiotypic phenotypes. In vitro admixture and in vivo challenge studies resulted in consonant differential suppression. Thus idiotypic regulation can mold the fine specificities of the protective immune response to S. mansoni at the clonal level and may provide an approach to optimize the expression and assessment of resistance.     

Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization

These studies address the hypothesis that Ag-bearing epidermal Langerhans cells migrate to the regional lymph node during contact sensitization and function as APC. Skin from C3H mice was grafted onto BALB/c nude mice, and 7 or 14 days later, the recipients were sensitized with FITC through the grafts. APC from lymph nodes draining the site of sensitization were capable of sensitizing C3H recipients to FITC. Because sensitization is MHC restricted, only cells reaching the lymph node from the grafted skin could have induced contact hypersensitivity in C3H mice. Examination of the FITC+ draining lymph node cells by immunofluorescence and immunoelectron microscopy demonstrated that all were Ia+, most were F4/80+, and some contained Birbeck granules. These studies demonstrate that Ia+, FITC+ cells from the skin, at least some of which are Langerhans cells, leave the skin after epicutaneous sensitization with FITC and participate in the initiation of the contact hypersensitivity response within the regional lymph node.     

In vitro generation of natural killer cells from SJL/J bone marrow precursors

The development of natural killer (NK) cells from undifferentiated bone marrow (BM) precursors of low-NK-reactive SJL/J mice was studied. Results indicate that BM cells of untreated mice are not able to generate NK effector cells in cultures supplemented with recombinant interleukin-2 (IL-2). On the other hand in the presence of IL-2, NK cells are generated in cultures of BM from mice pretreated with 5-fluorouracil (5-FU, 150 mg/kg iv 4 days before harvesting), a treatment which has been shown to eliminate more differentiated but spare less differentiated BM precursors. The 5-FU resistant BM progenitor is asialoGM1-, Thy.1+, Lyt.1- and Lyt.2-. The cells generated by culturing with IL-2 are asialoGM1+, Thy.1+, Lyt.5+, Lyt.1-, Lyt.2- and lyse only NK-susceptible targets. Generation of NK cells is blocked by addition of anti-IL-2 receptor (IL-2/r) antibodies. These studies demonstrate that it is possible to generate NK effectors from SJL/J BM cells by in vitro culturing with IL-2.     

Immune suppression with supraoptimal doses of antigen in contact sensitivity. I. Demonstration of suppressor cells and their sensitivity to cyclophosphamide.

Immunologic suppression was induced in a mouse model of contact sensitization to DNFB by using supraoptimal doses of antigen. In these studies, in vivo measurement of ear swelling as an indication of immunologic responsiveness correlated well with measurement of in vitro antigen-induced cell proliferation. This unresponsiveness was specific, since supraoptimal doses of DNFB did not interfere with the development of contact sensitivity to another contactant, oxazolone. The decrease in responsiveness is a form of active suppression, as lymphoid cells from supraoptimally sensitized donors transferred suppression to normal recipients. Furthermore, pretreatment with cyclophosphamide (Cy) reversed the suppression seen in supraoptimally sensitized animals but had no effect on the optimal sensitization regimen. These results indicate that supraoptimal doses of contactants can activate suppressor cells and that precursors of these cells are sensitive to Cy. Such suppressors regenerate within 7 to 14 days after Cy treatment. The ability of Cy pretreatment to affect supraoptimal sensitization without affecting optimal sensitization confirms other reports indicating that the observed results of Cy treatment depend critically upon the dose of antigen used.     

Effects of the desiccation on Biomphalaria tenagophila (Orbigny, 1835) (Mollusca) infected by Schistosoma mansoni Sambon, 1907

Specimens of Biomphalaria tenagophila exposed to miracidia of Schistosoma mansoni were submitted to different desiccation periods as follows: group I: 24 h after exposure, desiccated for 28 days; group II: after cercariae elimination, desiccated for 7 days; group III: 21 days after exposure, desiccated for 7 days; group IV: 14 days after exposure, desiccated for 14 days; group V: 7 days after exposure, desiccated for 21 days. From the obtained data it was verified that desiccation was not capable of interrupting the development of larvae of S. mansoni in mollusks. A delay in the development of S. mansoni larvae in groups I, III, IV and V was observed. A pause was verified in the development of S. mansoni larvae in groups II, III, IV and V. Some larvae, in groups I, III, IV and V, did not suffer as a result of desiccation and continued their development. Larvae in the cercariae stage were shown to be more sensitive to desiccation. It was possible to obtain clearing of mollusks infected by sporocysts II and cercariae using a period of 7 days of desiccation.     

The effect of anti-tumor necrosis factor (TNF)-α monoclonal antibody on allergic cutaneous late phase reaction in mice

Biphasic cutaneous reaction with peak response at 1 (early phase) and 24 to 48 hour (late phase)was elicited by epicutaneous challenge with antigen in actively and passively sensitized mice. Mice were actively immunized with dinitrophenylated (DNP) ascaris antigen and challenged with dinitrofluorobenzene (DNFB). Passively sensitization was carried out by the injection of monoclonal anti-DNP-IgE antibody into mice and challenge was elicited with DNFB. Prednisolone at doses of 3 to 10 mg/kg clearly inhibited both early and late phase reactions in either sensitized mice. Monoclonal anti-tumor necrosis factor (TNF)-α antibody inhibited the late phase cutaneous reaction in actively sensitized mice. Anti-interleukin-5 (IL-5) monoclonal antibody has no effect on both phase reactions in either actively and passively sensitized animals. These results indicate the possible participation of TNF-α in allergic cutaneous late phase reaction in actively sensitized mice.