Modulation of the immune system in the mouse. 2. Drug administration following antigen sensitization

The effects of administering drugs 2 days after antigen sensitization in a combined model of delayed type hypersensitivity (DTH) and humoral immunity (HI) in the mouse are described and compared with a previous study in which drugs were administered prior to sensitization using the same model.Low doses of cyclophosphamide (CY) administered after antigen sensitization were found to enhance DTH to methylated bovine serum albumin (MBSA). In addition, high doses of this drug suppressed DTH for 3 days after challenge, and then enhanced the response for a further 5 days. The HI response to sheep red blood cells (SRBC) was simultaneously suppressed by a range of CY doses.A number of different drug types demonstrated a similar pattern of dose-dependent DTH effects. However, unlike the previous study DTH enhancement was not confined to the alkylating agents. Either DTH enhancement or suppression was also observed after treatment with a number of other drugs.HI was suppressed by several drugs, in particular by a number of alkylating agents and anti-metabolites. Selective effects on one or other limbs of the immune response were observed.The mode(s) of action of drugs active in the present study are uncertain but possible explanations are discussed in terms of the elimination and subsequent recovery of specific cell populations.     

Long-lasting enhancement of the delayed-type hypersensitivity response to heterologous erythrocytes in mice after a single injection of cyclophosphamide.

Previous reports have indicated that cyclophosphamide (CY) treatment can enhance delayed-type hypersensitivity (DTH) reactions by abrogating suppressor T cell functions. Such findings have suggested that cells in the suppressor lineage may be particularly sensitive to this alkylating agent. The experiments reported here demonstrate that a single injection of CY before sensitization can induce a long-lasting state of enhanced DTH responsiveness to sheep red blood cells (SRBC) in mice. This enhancement required concurrent antigenic stimulation and appeared to be antigen-specific. Additionally, CY treatment of sensitized mice before the first antigenic challenge for DTH resulted in suppressed responses to that challenge, followed by enhanced DTH to subsequent challenge with the same antigen. The suppressed response was achieved with a lower dose of CY than the subsequent enhancement and also required concurrent antigenic stimulation. These results indicate that the effects of CY on both effector and suppressor mechanisms are critically dependent upon antigenic stimulation, and suggest that apparent suppressor sensitivity to CY may be a function of differential ability to recover from CY treatment in a context of antigenic stimulation.     

Effect of hydrocortisone, cyclophosphamide, azathioprine and methotrexate on cutaneous delayed and arthus hypersensitivity in the rat.

The effect of 4 immunosuppressive agents--hydrocortisone (HC), cyclophosphamide (CY), azathioprine (AZ) and methotrexate (MTX)--, on cutaneous delayed-type hypersensitivity (DTH) and Arthus reactions to the intradermal injection of ovalbumin (OA) in rats sensitized to OA in Freund's complete adjuvant (FCA) was studied. Multiple doses (daily for 4 days) were given either early, beginning on the day of sensitization, or late, beginning 9 days after sensitization. Single doses were given on the day of challenge with OA. All late multiple doses of drugs except HC depressed the DTH at 24 h in the following order of decreasing magnitude: MTX, CY, AZ. The DTH at 24 h was depressed by early multiple doses of MTX at all doses, by CY at all but the lowest dose, and by AZ at the intermediate dose; HC had no effect. When the drugs were given as single late doses, only CY at the lowest dose and MTX at the higher doses effectively depressed the DTH at 24 h. Increased Arthus reactions occurred after early and late multiple doses of HC and after a late single dose of CY at the highest dose. After late multiple doses the Arthus reaction was unaffected by either CY or MTX but was depressed by all doses of AZ. HC administered as 3 injections around the time of challenge markedly depressed the delayed and Arthus reactions. These results show that each of the 4 immunosuppressive drugs could depress DTH and Arthus reaction to OA, but the degree of depression varied with the time of drug administration relative to sensitization and challenge, and the dose of drug used. Histologic examination of skin test sites showed that an apparently negative reaction did not necessarily imply total absence of a cellular inflammatory response.     

Specific Suppression of Humoral and Delayed Hypersensitivity Responses by Cyclophosphamide in an Experimental Model of Autoimmunity

ABSTRACT: The aim of this report is to investigate the effect of cyclophosphamide (CY) in an experimental model of autoimmunity to rat male accessory glands. The results indicated that 100 mg/kg of this drug suppressed humoral immune response that persisted for at least 45 days when administered 3 days after the first immunization of rats with modified rat male accessory glands (MRAG) in complete Freund's adjuvant (CFA). Administration of the drug 3 days before ID injection of antigen caused a shorter suppression of antibody formation. Delayed type hypersensitivity (DTH) studied 13 days after the first immunization was suppressed only in the animals that were administered CY after the antigen. The specificity of the immunosuppression was studied in rats treated with CY after the first immunization with MRAG using aggregated human γ-globulin (AHGG) as an unrelated antigen. The studies demonstrated significant suppression of DTH (p < 0.005) and humoral immunity only against MRAG. On the contrary, the response to AHGG was not significantly modified.     

Local administration of cytostatic drugs enhances delayed-type hypersensitivity to Sendai virus in mice

Cytostatic drugs known to exert immunomodulatory activity were studied in a local treatment protocol as to their effect on development of cell-mediated immunity to Sendai virus in mice. Strong enhancement of delayed-type hypersensitivity (DTH) was observed after treatment with the active cyclophosphamide (CY) derivative Z 7557 or the plant alkaloid VP-16 at the site of sensitization. Optimal enhancement of DTH was obtained after priming with subimmunogenic doses of Sendai virus and treatment with low, virtually nontoxic drug doses. Immunoenhancement is ascribed to elimination of CY-sensitive suppressor cells from the draining lymph nodes. The relevance of our findings with respect to protective immunity is discussed.     

Opposite effects of cyclophosphamide pretreatment on tuberculin hypersensitivity during the course of sensitization of mice with Mycobacterium bovis BCG

The influence of cyclophosphamide (CY) pretreatment upon the development of tuberculin hypersensitivity has been studied during the course of infection of mice with BCG. An enhancement of the DTH response to BCG antigens occurred during the induction phase, whereas a depression of this response occurred at the peak and during the decay phase of sensitization. The development of the early DTH-promoting and of the late DTH-depressing effect of CY was favoured by the use of a supra-optimal dose of BCG. Both these effects were cell-dependent since they could be transferred adoptively to syngeneic recipient mice with sensitized lymphoid cells but not with specific immune sera. Pretreatment with CY favoured the emergence of cells capable of responding in vitro to BCG antigens in the draining lymph nodes of BCG-infected mice. No simple association however, exists between this in vitro lymphocyte transformation response and the DTH response.     

Kinetics of the relation between suppressor and effector mechanisms in contact sensitivity in the guinea-pig

Cyclophosphamide (300 mg/kg) given before or up to 2 days after sensitization, induces increased contact skin reactions at 8 days. Reactions were suppressed with cyclophosphamide (CY) given between 3 and 5 days after sensitization; reactivity returned on day 10. CY, given on days 6 to 8, only suppressed reactions when skin tests were made 4 days later. This temporary depression of contact sensitivity corresponds with the maximal reduction of peripheral blood lymphocytes. CY given 1-2 days after DNFB produced decreased T-cell proliferation in local lymph nodes 4 days after sensitization. CY given 3 days after DNFB produced maximal T-cell suppression on 5 day nodes. Massive increase in T-cell proliferation in 5 day nodes occurred when CY was given on the day of sensitization or the day before. Thus CY given around sensitization acts mainly on suppressor cells whereas given later, the action is principally on the effector functions.     

Inhibition of T suppressor cell function by local administration of an active cyclophosphamide derivative at the sensitization site.

In previous work, we have shown that local administration of active cyclophosphamide (CY) derivatives, or other cytostatic drugs, at the sensitization site induced a similar immunopotentiation to that of systemic CY. Since it is well documented that CY can inhibit suppressor cells, it was proposed that immunoenhancement by locally administered drugs might be based on a similar principle. The objective of the present study was to test this hypothesis, using an experimental model of Ts mediated suppression of delayed type hypersensitivity to sheep erythrocytes. In this model, mice are made tolerant to sheep erythrocytes by i.v. injection of a high dose of sheep erythrocytes. Local treatment of sheep erythrocytes-tolerant mice with the active CY derivative Z7557 at the site of attempted sensitization reversed suppression in a dose-dependent manner. Local treatment with the cytostatic drug etoposide (VP-16) and systemic CY treatment were also effective. In transfer experiments, the function of afferently acting suppressor cells was blocked by local treatment with Z7557 or systemic CY. These data support the concept of anti-suppressor cell activity of locally administered cytostatic drugs. As with CY, the pharmacological basis of this effect remains to be determined.     

Alterations in dendritic cell phenotype and function associated with immunoenhancing effects of a subcutaneously administered cyclophosphamide derivative.

A single systemic dose of cyclophosphamide (CY) has been shown to enhance cellular immunity in a variety of antigen models. The immunoenhancing effects of CY have been attributed to its ability to selectively abrogate suppressor cell function. Previous studies from our group have demonstrated that local administration of distinct cytostatic drugs at the sensitization site can induce a similar enhancement of delayed-type hypersensitivity as systemic CY, with the obvious advantage of avoiding systemic side-effects. In the present study we investigated the effects of local administration of an optimally immunopotentiating dose of the active CY-derivative Z 7557 and, in selected experiments, of etoposide (VP-16) and systemic CY on mononuclear cells in draining lymph nodes. Whereas CY caused a long-lasting and marked depletion of B-cell areas, locally administered Z 7557 and VP-16 relatively spared B cells and even induced an increase in B- and T-cell numbers in (keyhole limpet haemocyanin-) sensitized mice. At Day 4 the CD4/CD8 ratio was slightly reduced in drug-treated mice. Interestingly, drug treatment reduced the proportion of interdigitating cells staining with the monoclonal antibodies NLDC-145 and MIDC-8. Upon isolation, dendritic cells (DC) from sensitized, Z 7557-treated mice showed longer dendritic protrusions and an enhanced accessory cell function compared to DC from saline-treated controls. These findings suggest that immunoenhancing effects of cytostatic drugs may occur via an effect on DC.     

Immunomodulatory properties of monoclonal anti-Thy-1.2 antibody in vivo

Monoclonal mouse anti-Thy-1.2 antibody of IgG3 isotype (MTA) inhibits in a significant and long-term manner the regional graft-versus-host reaction (GVHR) when administered to donors as well as to recipients. The process of sensitization in the reaction of delayed type hypersensitivity to SRBC (DTH) is, on the other hand, suppressed only if MTA is administered immediately prior to sensitization. If the time interval between the injection of MTA and of SRBC is extended, the resulting DTH is stimulated. Similarly, the titers of total Ig against SRBC are decreased only if MTA is administered shortly before immunization. Contrasting effects of MTA on the production of IgM and IgG were observed. While the production of IgG was inhibited even after the time interval between the administration of MTA and immunization had been increased, the production of IgM antibodies was stimulated. In recipients of allogeneic Sarcoma I treated with MTA, the primary growth of the tumor is enhanced and the percentage of permanent progressors is increased. Mechanisms of MTA activity are discussed.     

Effects of contact sensitization and delayed hypersensitivity reactions on immune responses to non-related antigens. Modulation of Immune responses.

Guinea pigs were immunized intracutaneously into the ears with sheep red blood cells (SRBC). Application of a sensitizing dose of the contact allergen dinitrochlorobenzene (DNCB) onto the same ears was shown to suppress or enhance the humoral response to SRBC depending on the time of application. When guinea pigs were sensitized to a contact allergen, application of a sensitizing dose of a non-related allergen on the same ears either had no effect or caused a clear enhancement of the development of delayed type hypersensitivity (DTH). Strongest enhancement was found when both sensitizations were performed on the same day. Further experiments on the effects of a concomitant DTH reaction elicited at the site of application of a contact allergen showed a strong potentiation of DTH when B-cell suppression was minimized by pretreatment with cyclophosphamide (CY). It was considered that CY-DTH-immunopotentiation might be a useful tool for achieving a higher level of sensitivity after epicutaneous sensitization.     

The effect of immunomodulating treatment on cutaneous delayed-type hypersensitivity in MRL lpr/lpr mice

An autoimmune disease in MRL lpr/lpr (MRL/l) mice is associated with a plethora of T-cell abnormalities, one of them being impaired delayed-type hypersensitivity (DTH). Three immunomodulating drugs, cyclophosphamide (Cy), cyclosporin A (CsA) and LS 2616, all with beneficial effects on autoimmune diseases, have been examined with regard to their potential effects on DTH in female MRL/l mice and in healthy control mice. All three drugs, given as a single dose at the time of sensitization, increased DTH reactivity of oxazolone in healthy control mice, while only two of them, Cy and LS 2616, significantly augmented the defective DTH response in MRL/l mice.     

Regulation of delayed-type hypersensitivity. III. Effect of cyclophosphamide on the suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

A previous study (Eur. J. Immunol. 1977. 7: 714) has shown that mice injected intravenously (i.v.) with 4 x10(9) sheep red blood cells (SRBC) produce cells which suppress delayed-type hypersensitivity (DTH). These suppressor cells are theta-positive, antigen-specific and act via a soluble factor which does not bear immunoglobulin determinants (Eur. J. Immunol. 1978. 8: 168). The present paper demonstrates that these suppressor cells are inhibitable by cyclophosphamide (CY). Mice injected with graded amounts of CY two days prior to SRBC injection, showed maximum augmentation of DTH at 200 mg/kg body weight, a dose which completely suppressed the appearance of splenic plaque-forming cells (PFC) to SRBC. In contrast, lower doses of CY enhanced both DTH and PFC responses. Time course studies showed that CY inhibited the precursors of suppressor cells and had little or no effect on suppressor cells which have already encountered antigens. This was further confirmed by passive transfer studies which showed tha- suppressor cells were inhibited if CY was administered at the same time or 2 days before SRBC injection, but were not affected if CY was given after antigen stimulation. Direct evidence for the effect of CY on suppressor cells was obtained by cell fractination with a Ficoll density gradient. The denser suppressor cell population was absent from the spleens of mice treated with 200 mg/kg of CY 2 days before i.v. injection with 1 x 10(9) SRBC.     

Langerhans' cells in the murine oral mucosa in the inductive phase of delayed type hypersensitivity with 1-chloro-2, 4-dinitrobenzene

We created a murine model of delayed-type hypersensitivity (DTH) to 1-chloro-2, 4-dinitrobenzene (DNCB). Using this murine model, we compared oral mucosal sensitization and skin sensitization for the difference in reaction during the elicitation phase. Evaluation of sensitizability, using the mouse ear swelling test (MEST) after oral mucosal or skin sensitization, showed that the ear swelling response peaked 24 h after challenge. The optimal induction concentration was 1.0% in both oral mucosal and skin sensitization, resulting in a positive reaction rate of 100%. However, the ear swelling response 24 h after challenge with the optimal concentration of DNCB (1.0%) was significantly lower in oral mucosal than in skin sensitization. We compared the oral mucosal and skin sensitization sites for the number of Langerhans' cells (LC) and the antigen-presenting capability in the induction phase. The numbers of F4/80+ major histocompatibility complex (MHC) class II+ LC before induction did not differ significantly between the oral mucosa and the skin. After induction, F4/80+ MHC class II+ LC increased in number, but the increase was significantly smaller in the oral mucosa than in the skin. MEST on anti-CD86 antibody-administered mice showed that ear swelling was similarly suppressed after oral mucosal or skin sensitization. In murine models of DTH after oral mucosal sensitization, the number of F4/80+CD86+ LC increased after induction, but the increase was significantly smaller than that in murine models of DTH after skin sensitization. This study showed that, in murine models of DTH, oral mucosal sensitization elicited a weaker reaction than skin sensitization. This was presumably because oral mucosal sensitization induced fewer LC, resulting in lower antigen-presenting capability.     

The local anti-inflammatory activity of rimexolone (Org 6216) in fibrin-induced monoarticular arthritis and adjuvant-induced arthritis

The effects of a number of steroids administered intra-articularly in a chronic model of fibrin-induced monoarticular arthritis in the rabbit have been investigated. Org 6216, hydrocortisone acetate, prednisolone tertiary butyl acetate and triamcinolone hexacetonide each suppressed the joint swelling produced 14 days after antigen challenge. These anti-inflammatory effects lasted for at least 7 days. Hydrocortisone semisuccinate was inactive in this model.In addition, the effects of the same compounds and several other anti-inflammatory steroids and indomethacin administered locally with adjuvant was assessed on the resultant paw oedema produced in the rat. The local anti-inflammatory activity, the duration of action and the systemic effects of these drugs varied considerably and only Org 6216, hydrocortisone acetate, prednisolone tertiary butyl acetate and indomethacin produced anti-inflammatory effects throughout the 4 days of the experiments and were devoid of significant adrenolytic and thymolytic activity.     

Murine delayed-type hypersensitivity granuloma: an improved model for the identification and evaluation of different classes of anti-arthritic drugs

The present study examined the effects of five different classes of anti-inflammatory/immunoregulatory drugs using a mouse model of mBSA-induced delayed-type hypersensitivity granuloma (DTH GRA) to measure immune-mediated chronic inflammatory tissue formation. The compounds were administered orally daily following induction of DTH GRA (days 0 to 4); granulomata were quantitated gravimetrically on day 5. NSAIDs, with the exception of flurbiprofen, showed little activity in comparison with the steroids dexamethasone (1-3 mg/kg/day, orally) and prednisolone (3-10 mg/kg/day, orally), which caused significant suppression of DTH GRA tissue (65-76% and 26-68%, respectively). The "immunoregulatory" compounds levamisole and D(-)penicillamine were inactive, whereas cyclophosphamide (5-50 mg/kg/day, orally) reduced the response by 24-83%. The "interferon alpha-inducers" Tilorone, U-54,461, and U-56,499 were also potent inhibitors of the DTH GRA response; U-54,462, a weak interferon alpha-inducer, was inactive. Cyclosporin A (50-100 mg/kg/day, orally) suppressed DTH GRA most effectively when administered on days 3 and 4 (66% and 97%) of the five-day granuloma response (treatment was ineffective when given on days 1 and 2). We conclude that the DTH GRA response described above may be useful for evaluating different types of unique therapeutic agents that are effective in the treatment of chronic immuno-inflammatory disease such as rheumatoid arthritis.     

Establishment and comparison of delayed-type hypersensitivity models in the B6C3F1 mouse

The objective of these studies was to establish and compare delayed-type hypersensitivity (DTH) models, using keyhole limpet hemocyanin (KLH), sheep red blood cells (SRBC), and Candida albicans as sensitizing antigens, for their capability to assess a DTH response (utilizing footpad swelling as the endpoint) with minimal confounding factors resulting from antigen-specific antibody (Ab) production. The key elements of the DTH are the sensitization dose, time interval between sensitization and challenge [i.e. the challenge interval (CI)], and the challenge dose. Models were established by first determining the challenge dose, or the amount of antigen that produced no greater footpad swelling 24-h post-injection than the trauma induced by injection of physiological saline. Time-course studies determined the CI that produced a peak response for each antigen. Dose-response sensitization studies were conducted to determine the optimum sensitization concentration (i.e. maximum footpad swelling with minimal impact by antigen-specific Ab production). Footpad swelling decreased dose-responsively with increasing KLH sensitization concentration and corresponded to a dose-responsive increase in KLH-specific Ab levels. In the SRBC model, footpad swelling decreased at the high dose (1?×?10⁹ SRBC/mouse), and a corresponding increase in SRBC-specific Ab was observed at this dose level. A dose-responsive increase in footpad swelling was observed in the C. albicans model up to 3?×?10⁷ organisms/mouse, while antigen-specific antibody levels were not different from background (unsensitized) levels following sensitization with any concentration of C. albicans (up to 1.2?×?10⁸ organisms/mouse, the highest concentration tested). Finally, each model was evaluated for its ability to detect immunosuppression following exposure to benzo[a]pyrene (B[a]P), with the C. albicans model demonstrating greater sensitivity than the other models. These results indicate that, of the three models examined here, the C. albicans DTH model may be the most appropriate model for evaluating effects on cell-mediated immunity when conducting immunotoxicological investigations.     

白细胞移动抑制因子(LMIF)释放的测定及条件性抑制LMIF的作用

目的选用与迟发过敏（ＤＴＨ）反应密切相关的白细胞移动抑制因子（ＬＭＩＦ）的体外测定方法，分析在条件性免疫抑制反应中ＬＭＩＦ活性的变化。方法ＬＭＩＦ测定是以间接毛细管方法结合分光光度计，测定游出细胞的吸光度（Ａ值）。在条件性反应训练和建立ＤＴＨ反应模型的第７天，取耳称重，同时取小鼠肠系膜淋巴结细胞，制备ＬＭＩＦ。结果ＬＭＩＦ在条件反应组的作用明显减弱（Ａ值升高），与不经过条件性训练的对照组相比，差异有非常显著意义（Ｐ＜０．０１，方差分析，ｎ＝７），但与非条件反应组相比没有差异，说明已形成了条件性抑制ＬＭＩＦ的反应。结论本工作建立了稳定的ＬＭＩＦ吸光度检测法，建立了环磷酰胺条件性抑制ＬＭＩＦ的模型。为深入分析条件性免疫抑制反应的血清中的介导物质提供了一个方便的体外检测手段     

Potentiation of delayed-type hypersensitivity by pertussigen or cyclophosphamide with release of different lymphokines.

The effects of lymphokine production of two agents known to potentiate delayed-type hypersensitivity (DTH), pertussigen (pertussis toxin) (PT) and cyclophosphamide (CY) have been investigated. These two agents were administered to immunized mice. Subsequently, lymph nodes and spleen cells were exposed to specific antigen in vitro. The resulting culture supernatants were assayed for the presence of lymphokines. Only supernatants of cells from the mice given PT contained appreciable quantities of interferon-gamma (IFN-gamma) and stimulated cells of the monocyte-like WEHI-265 cell line to produce procoagulant activator and plasminogen activator. On the other hand, CY was more effective than PT on the production of interleukin-3 (IL-3). Both adjuvants had small enhancing effects on the production of interleukin-2 (IL-2). With either adjuvant, the cell populations induced had a similarly enhanced capacity to transfer DTH. These results demonstrate that the capacity of cells to transfer DTH does not necessarily correlate with their release of particular lymphokines. The potentiation of DTH by cyclophosphamide did not depend on significantly enhanced generation of IFN-gamma, procoagulant activator, or plasminogen activator. The amount of IFN-gamma in the culture supernatants correlated with their capacity to produce procoagulant activator and plasminogen activator, whereas the amount of IL-2 and IL-3 did not.     

Kinetics of the effect of a single dose of cyclosporin-A on antibody and cell mediated immune responses in the guinea pig

The effect of 100 mg/kg cyclosporin-A (CS-A), given as a single dose either before or after immunization, on antibody levels and skin test reactivity was investigated. CS-A was found to suppress both primary and secondary anti-hapten and anti-carrier IgG1 and IgG2 antibodies. However, CS-A was also capable of inducing enhanced anti-hapten antibodies. CS-A showed a similar effect on contact sensitivity reactions to 2,4-dinitrofluorobenzene (DNFB) as has been shown for cyclophosphamide (CY) in that, given before sensitization, skin reactions were enhanced, whereas given after sensitization they were suppressed. However, the effect of CS-A on the T-cell proliferation in the lymph node, draining the site of sensitization to DNFB, differed from that of CY. Although CS-A induced a depression of T-cell proliferation, this suppression was more prolonged than that found in CY treated animals. Also, these draining lymph nodes never showed increased T-cell proliferation as did those in animals treated with CY before sensitization. This work demonstrates that a single dose of CS-A can both suppress or enhance antibody production and delayed hypersensitivity. The timing of the dose of CS-A in relation to the time of primary immunization is important. However, both IgG1 and IgG2 primary and secondary antibody responses can be altered. Comparison with the effect of CY on antibody levels and contact sensitivity would indicate that in some ways CS-A reacts similarly to antimitotic agents and in other ways is different.