Immunosuppressive antimetabolites inhibit induction of contact hypersensitivity while lymphoablative drugs also prevent its expression

ARTICLE

Auteur(s) : Laurence QUÉMÉNEUR^1,3, Marie-Cécile MICHALLET^1,3, Carole FERRARO-PEYRET¹, Pierre SAINT-MÉZARD², Josette BENETIÈRE², Marie-Thérèse DUCLUZEAU², Jean-François NICOLAS², Jean-Pierre REVILLARD^1,4

¹ Laboratory of immunopharmacology and
² Laboratory of clinical immunology, INSERM U503, Claude Bernard University, 21, avenue T. Garnier 69365 Lyon Cedex 07, FRANCE.
⁴ Deceased on 2 June 2003.

Article accepted on 5/8/03

Contact hypersensitivity (CHS) is a T cell-mediated cutaneous inflammation elicited by epicutaneous exposure to haptens in sensitized individuals. The CHS response develops in two distinct phases. During sensitization, Langerhans cells process haptenized peptides and migrate from the epidermis to the skin draining lymph node [1, 2], where specific T cells are primed. Recent studies have outlined the important role of cytokines [3] and chemokines including CCL3 (MIP1-α), CCL4 (MIP-1β) [4] and CCL21 [5], and that of costimulatory signals in the sensitization phase [6-9]. The elicitation phase of CHS is induced by reexposure of the same hapten at a remote site skin. This leads to the rapid recruitment and activation of specific T cells, and to the constitution of a local inflammatory response. Using dinitrofluorobenzene (DNFB) as the sensitizing hapten in mice, the effector cells have been characterized as IFN-γ producing CD8⁺ T cells [10]. Their cytotoxic activity towards keratinocytes by either Fas or perforin pathways was shown to be mandatory for full expression of the CHS expression [11, 12]. Conversely, CD4⁺ T cells exert a regulatory activity in this model [10, 13, 14].
The pharmacological control of CHS is an important clinical issue. A great number of studies have dealt with the effect of topical or systemic administration of corticosteroids and calcineurin inhibitors such as cyclosporine A or tacrolimus (FK506). By contrast, most studies with other immunosuppressive drugs were performed in the 60s long before acquisition of the present knowledge on the mechanisms of CHS. Interestingly, before the recent identification of regulatory T cells, several reports dealt with the potentiating activity of pretreatment with cyclophosphamide (CYP) [15, 16] or administration of low doses of CYP in CHS transfer experiments [17].
The present study was designed to investigate the action of two groups of immunosuppressive molecules which were shown in other models either to interfere mostly with dividing lymphocytes, or to deplete lymphoid cells in a cell cycle independent manner. We postulated that induction of CHS would require T cell proliferation whereas the development of the inflammatory reaction at the time of challenge would be resistant to cell cycle dependent agents. To this aim we applied the drugs for a brief period during induction or during challenge of CHS to DNFB in mice. In this study, we selected five molecules characterized by different chemical targets and mechanisms of action. Three of them belong to the group of antimetabolites, i.e. drugs which interfere with critical enzymatic activities in the biosynthetic pathways of purine or pyrimidine nucleotides. Mycophenate mofetil (MMF) the prodrug of Mycophenolic acid (MPA), a specific inhibitor of type II inosine monophosphate deshydrogenase, is currently used in clinical organ transplantation [18] and is being evaluated in autoimmune and inflammatory diseases. Methotrexate (MTX) is efficient in patients with rheumatoid arthritis and other autoimmune disorders. MTX targets several folate-dependent enzymes and exerts pleiotropic effects. Its anti-inflammatory activity was demonstrated in numerous experimental models: it is mostly accounted for by the production of adenosine [19] which may also contribute to immunosuppression by suppressing NF-κB activation [20]. However, the immunosuppressive activity of MTX predominantly involves the blockade of thymidylate synthase resulting in a cytostatic or cytotoxic effect, restricted to activated T cells in vitro [21] and in vivo [22]. We also evaluated a specific inhibitor of thymidylate synthase, 5-fluorouracil (5-FU), which is extensively used in cancer chemotherapy but not in immunological disorders. In this study we compared these three antimetabolites with two cytotoxic drugs: CYP and mitoxantrone (MXN). CYP is an alkylating agent used in several autoimmune or inflammatory diseases. MXN is an intercalating agent used in B cell lymphomas and recently applied to multiple sclerosis [23]. These two drugs will be referred to as lymphoablative because, as shown in this study, they induce a profound T and B cell depletion upon a single injection in mice.

Materials and methods

Mice and reagents

Female C57BL/6 mice were obtained from IFFA Credo (L'Arbresle, France), maintained under specific pathogen-free conditions, and used at 7-8 weeks of age.
DNFB, MTX, CYP and MPA were obtained from Sigma (St Quentin Fallavier, France). MXN was purchased from Wyeth-Lederle (Paris, France) and 5-FU from Teva^® Pharma (Courbevoie, France). MMF was obtained from Roche Bioscience (Palo Alto, CA). The volume of the drug given to each animal was adjusted according to body weight. MTX, 5-FU, MXN and CYP were diluted in PBS and injected in the i.p. cavity. MMF was resuspended in a vehicle buffer (0.5% carboxymethyl cellulose, 0.9% NaCl, 0.4% Tween 80 and 0.9% Benzyl alcohol) and was given by oral gavage. Control mice received vehicle only.

Isolation of splenocytes and lymph node cells

Spleen and mesenteric lymph nodes from C57BL/6 mice were removed. Single cell suspensions were made by mashing spleen or lymph nodes on a nylon cell strainer (100 µm). Cells were resuspended in complete RPMI 1640 medium supplemented with 10% FCS, 2 × 10 ^– 5 M β2-Mercaptoethanol, 2 mM L-glutamine and antibiotics (Penicillin 100 U/ml, Streptomycin 100 µg/ml). Viable cells were counted by trypan blue dye exclusion.

Lymphocyte subsets analysis by FACS

After a wash in PBS containing 2% BSA and 0.2% NaN3 (PBS/BSA/Azide), cells (5 × 10⁵) were incubated with 5 µl of FITC-conjugated mAb for 30 min at 4°C. After washes, cells were resuspended in PBS/BSA/Azide buffer and analyzed by flow cytometry, using a FACScalibur and the CellQuest software (Becton Dickinson, Pont de Claix, France). FITC-conjugated anti-CD4, anti-CD8 and anti-B220 were obtained from Becton Dickinson.

Cell cultures

Lymph node cells (2 × 10⁵ per well) were either activated for 48 h with concanavalin A (Con A, 5 µg/ml) (Sigma) and IL-2 (20 U/ml) (Chiron, Suresnes, France) or supplemented with IL-7 (12.5 ng/ml) (Peprotech, TEBU, Le-Perray-en-Yvelines, France). Cells were then treated with immunosuppressive drugs for 24 h and the percentage of apoptotic cells was determined by flow cytometry. Briefly, cells were resuspended in annexin V-binding buffer containing FITC-conjugated annexin V for 15 min following instructions of the manufacturer (Bender Medsystems, Austria). Propidium iodide (1 µg/ml) was then added and cell suspensions were immediately analyzed by flow cytometry. For proliferation assay, cells were pulsed for 30 min with ³[H]thymidine (Amersham France SA, Les Ulis, France) at 0.5 µCi/well. ³[H]thymidine uptake was measured using a Packard direct counter (Packard, Meriden, CT) after harvesting.

Contact hypersensitivity

The CHS assay was performed as previously reported [12]. DNFB was diluted in acetone/olive oil (4:1) immediately before use. C57BL/6 mice were sensitized by topical application of 0.5% DNFB (20 µl) to shaved abdominal skin. Five days later, one side of the left ear was challenged with 10 µl of 0.2% DNFB (a nonirritant concentration) and the right ear with the solvant alone. Ear thickness was monitored using a micrometer (J15; Blet SA, France) before challenge and every day after challenge. The ear swelling was calculated as [(T-T₀)_{left ear}] - [(T-T₀)_{right ear}], where T and T₀ represent values of ear thickness after and before challenge, respectively. It has been shown repeatedly in previous experiments that topical application of 0.2% DNFB in unsensitized mice did not induce more ear swelling than the solvent alone in sensitized mice. In each experimental group, some mice were sacrified at different time intervals after DNFB challenge for histological and PCR analysis.

RNA extraction and reverse transcription PCR analysis of CD8 and IFN-γ mRNA

The technique has been previously reported [12]. Briefly, at different time intervals after challenge, ear samples were collected and frozen in liquid nitrogen. Total RNA was extracted using an RNA PLUS Isolation kit (Quantum, Appligene, Illkirch, France). After Dnase I treatment, 1 µg of total mRNA was reverse transcribed using oligo-dT15 primers and Superscript II RT (90 min, 37°C, GIBCO BRL). The amount of RNA to be used for detection was normalized using the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) as reference. The cDNA obtained was amplified using different sets of primers: for HPRT (5' primer, 5'-GTA ATG ATC AGT CAA CGG GGG AC-3'; 3' primer, 5'-CCA GCA AGC TTG CAA CCT TAA CCA-3'), for CD8 (5' primer, 5'-AGG ATG CTC TTG GCT CTT CC-3'; 3' primer, 5'-TCA CAG GCG AAG TCC AAT CC-3') and for IFN-γ (5' primer, 5'-GCT CTG AGA CAA TGA ACG CT-3'; 3' primer, 5'-AAA GAG ATA ATC TGG CTC TGC-3'). The amplifications were carried out with 29 cycles for HPRT and 33 cycles for CD8 and IFN-γ (1 min at 94°C, 1 min at 60°C and 1.5 min at 72°C). The PCR products were analyzed on 1.5% agarose gel.

Migration of Langerhans cells to the draining node

The assay was performed as previously described [2]. Briefly, both ears were painted with 10 µl of 1.5% FITC. Twenty-four hours after exposure to FITC, mice were sacrified, cervical lymph nodes were removed, tissue was teased and cells were filtered through a 100 µm cell strainer. Single cell suspensions from inguinal lymph nodes were used as control. Single cell suspensions were washed and layered onto a metrizamide gradient (14.5% in RPMI 1640, 7.5% FCS) and centrifuged for 10 min at 600xg. Cells at the interface were collected, washed and labeled with biotinylated anti-MHC class II plus streptavidin-conjugated Cyanin mAbs (Pharmingen, Becton Dickinson) and analyzed by flow cytometry. Cells labeled with both FITC and Cyanin were quantified as migrating skin dendritic cells.

Results

In vitro lymphocyte apoptosis and inhibition of T cell mitogenic responses

The selected molecules were added to lymph node cells for 24 h and the percentage of apoptotic cells was determined by annexin V binding. Such cell suspensions contained about 40% CD4⁺ T cells and 15% CD8⁺ T cells (data not shown). Only MXN triggered apoptosis of these non-activated lymph node cells in a dose-dependent manner (Fig. 1A). By contrast not only MXN but also 5-FU, MTX and to a much lesser extent MPA were found to be cytotoxic on Con A-activated lymph node cells (Fig. 1A). Such cell suspensions contain nearly exclusively cycling T cells with about 70% CD4⁺ and 30% CD8⁺ with an intermitotic interval of 6-8 h (data not shown). The four drugs dose-dependently inhibited ³[H]-thymidine incorporation into Con A-activated T cells (Fig. 1B). This effect is likely to be associated with apoptosis as regards MTX, 5-FU and MXN. As regards MPA, the strong inhibition of T cell proliferation contrasted with a borderline cytotoxic activity, suggesting that MPA had a genuine antiproliferative effect. Note that in contrast to antimetabolites, both resting and cycling lymphocytes were sensitive to apoptosis induced by MXN. CYP is a prodrug which must be metabolized into 4-hydroperoxycyclophosphamide by hepatocytes in vivo to become active, therefore its effect on lymphocytes in vitro was not analyzed. Altogether, these results suggest that MTX and 5-FU are cytotoxic for dividing T cells whereas MXN is toxic for both dividing and non-activated T cells. MPA is mostly anti-proliferative and displays a borderline cytotoxic effect on activated T cells only.

T- and B-cell depletion in vivo induced by MXN and CYP, but not antimetabolites

We next asked whether the capacity of the lymphoablative drugs, but not antimetabolites, to induce apoptosis of non-activated lymphocytes in vitro could be associated with a cell depleting effect in murine lymphoid organs. To this end, C57BL/6 mice were injected with PBS, MTX (7 mg/kg/day), 5-FU (40 mg/kg/day) for 2 days or only once with CYP (300 mg/kg) and MXN (10 mg/kg). MMF (100 mg/kg/day) was administered by oral gavage for 2 days. The doses of 5-FU and MTX were determined by preliminary experiments of dose escalation ([22] and data not shown) showing that a daily administration of the selected dose over a period of one week did not induce obvious toxicity. The dose of MMF is in agreement with previous studies from our group [24] and others [18]. The dose of MXN was established by dose escalation and evaluation of one month survival after a single injection (data not shown). CYP was tested at doses of 100, 200 and 300 mg/kg in preliminary experiments. Doses of 200 mg/kg as single injection induced significant B but little T cell depletion [25]. Therefore the dose of 300 mg/kg was selected. This dose was not toxic but sufficient to induce T cell depletion as shown below. Forty-eight hours after the last drug administration, spleen and lymph nodes were removed, cells were isolated and viable cells were counted. Administration of MTX, 5-FU or MMF had no effect on lymphocyte counts, whereas CYP and MXN reduced cell number in both spleen and lymph nodes compared to mice injected with PBS (Fig. 2A). In order to investigate which lymphoid subpopulations were affected by CYP or MXN treatment, spleen and lymph node cell suspensions were analyzed using T- and B- cell markers. Spleens of PBS-treated mice contained 43 × 10⁶ CD4⁺, 23 × 10⁶ CD8⁺, 152 × 10⁶ B220⁺ cells as shown in Fig. 2B. The spleen cell number decreased to 34 × 10⁶ CD4⁺ T cells, 18 × 10⁶ CD8⁺ T cells and 40 × 10⁶ B220⁺ cells in mice treated with MXN. CYP induced an even stronger depletion of T and B splenocytes with 10 × 10⁶ CD4⁺, 8 × 10⁶ CD8⁺, 11 × 10⁶ B220⁺ cells (Fig. 2B). In lymph nodes, both CYP and MXN induced a 7-fold decrease in CD4⁺ and CD8⁺ T cells and a 23-fold decrease in B cells (Fig. 2B). Therefore, CYP and MXN affected the three lymphoid subpopulations, suggesting that these two drugs induced a T- and B-cell depletion demonstrable as soon as 48 h after a single injection.

Inhibition of induction but not expression of CHS by MTX, 5-FU and MMF

In order to evaluate the effect of antimetabolites on CHS, drugs were administered during the induction or the expression phase. Sensitized mice developed a reaction upon challenge with DNFB which peaked at 24 or 48 h and faded away beyond 72 h. Administration of MTX or 5-FU during the induction phase dramatically reduced the reaction as compared to the untreated sensitized challenged mice (Fig. 3A). Conversely, injection of MTX or 5-FU 4 h before challenge and once a day thereafter had no effect on the inflammatory response (Fig. 3B). Oral gavage with MMF during the induction phase completely inhibited the CHS reaction at 24 h and ear edema was reduced by 50% compared to vehicle-treated mice at 48 h but remained slightly above control (Fig. 3C). Similar to what was observed with MTX and 5-FU (Fig. 3B), administration of MMF during the elicitation phase starting 4 h before challenge did not alter the ear swelling at the peak of reaction (24 h) but slightly increased the edema at 48 h (Fig. 3D).

Inhibition of both induction and elicitation of CHS by CYP and MXN

We next investigated the effect of lymphoablative drugs on CHS. A single bolus injection of CYP (300 mg/kg) was administered 12 h before the time of induction or elicitation of CHS or one bolus of MXN (10 mg/kg) 4 h before induction or challenge (Fig. 4). As with antimetabolites, induction of the reaction was completely abrogated by administration of lymphoablative drugs (Fig. 4A). More surprisingly, the reaction was also inhibited when CYP or MXN were injected at day 5, immediately before challenge (Fig. 4B). CYP completely reduced the reaction to the level of the unsensitized challenged mice and MXN induced an inhibition of 65% of the reaction at the peak of edema, 24 h after challenge, and ear swelling remained reduced even 96 h after challenge.

Histological analysis of the CHS after treatment with immunosuppressive drugs

In order to confirm the effect of lymphoablative and antimetabolite drugs during the elicitation phase of the CHS, we evaluated the pathological changes occurring in the skin. Histological analysis of challenged sites showed that CHS in PBS-treated mice was associated with vascular enlargement, dermal edema, and infiltration by mononuclear cells (Fig. 5). These typical histological features of CHS were not modified in mice treated with antimetabolites from the day of challenge. Conversely, none of the characteristic pathological changes were observed in CYP- or MXN-treated mice, and challenged sites had the same histological aspect as in naive unsensitized mice.

Effect of immunosuppressive drugs on CD8⁺ T cell infiltration and IFN-γ expression

In this model, the elicitation phase is characterized by an early recruitment (6 h) of cytotoxic CD8⁺ T cells which secrete IFN-γ and induce apoptosis of keratinocytes in challenged skin [11]. The regulatory CD4⁺ T cell population migrate later (18 h) to the inflammatory site and resolution of skin inflammation is initiated when those cells penetrate the lesion. We therefore examined the presence of CD8 and IFN-γ mRNA in the ear. Ear samples from sensitized drug-treated or untreated mice were collected 24 h after DNFB challenge and subjected to mRNA extraction and semiquantitative RT-PCR analysis using HPRT mRNA as internal standard. CD8 and IFN-γ mRNA were not detected in ear skin of unsensitized DNFB challenged mice. Twenty-four hours after DNFB challenge, in PBS-treated challenged mice, CD8 and IFN-γ mRNA were detected, confirming that activated, fully differentiated cytotoxic CD8⁺ T cells infiltrated the challenged skin. Administration of antimetabolites or lymphoablative drugs during the induction phase inhibited the recruitment of CD8⁺ T cells in the ear skin (Fig. 6A). Conversely, in keeping with the measurement of ear swelling (Fig. 3 and  4), only lymphoablative drugs but not antimetabolites injected during the expression phase suppressed the infiltration of CD8 in the challenged ear skin (Fig. 6B).

Lack of interference of immunosuppressive drugs with dendritic cell maturation and migration to the draining lymph node

In order to check if the immunosuppressive activity of the drugs used in this study involved dendritic cells rather than T lymphocytes, FITC was used as hapten and applied to the ear skin (Table I). Then the draining lymph nodes were harvested 24 h later and fluorescent MHC class II positive cells were counted by FACS analysis. None of the tested drugs was found to interfere with dendritic cells migration to the lymph nodes. Furthermore the level of MHC class II expression by these cells was not decreased after administration of antimetabolites or lymphoablative drugs (Table I).

Discussion

In this study we have assessed how different immunosuppressive or lymphoablative drugs could interfere with the induction or the expression of CHS. We show here that two extensively used immunosuppressive drugs, MMF, a purine synthesis inhibitor, and MTX, an inhibitor of folate dependent enzymes, both suppress the induction of CHS but do not significantly alter its expression when administered immediately before challenge. 5-FU has the same effects as MTX in this model. Inhibition of CHS induction by these drugs is likely to be accounted for by their capacity to inhibit cell proliferation or to induce apoptosis of cycling but not resting T cells in vitro. These results are in agreement with our recent study showing that purine and pyrimidine nucleotides control cell cycle, proliferation and survival of human primary activated T lymphocytes [26]. Pioneering studies by Turk and coworkers [27] elegantly demonstrated, by pulse-chase administration of radioactive thymidine and autoradiography of the ear draining lymph node after sensitization with oxazolone, that the induction phase was characterized by the enlargement of the deep cortex with extensive proliferation of blast cells. Since the hapten binds to lysine residues of many different peptides, CHS is the prototype of a highly polyclonal yet specific T cell response. In vitro, depending on the drug concentration, MTX either inhibits proliferation or induces apoptosis of cycling human T cells [21, 26]. A similar effect was observed here with Con A-activated mouse T cells. Furthermore apoptosis of lymph node T cells following injection of a bacterial superantigen was demonstrated in mice treated with MTX or another thymidylate synthase inhibitor [22]. These data are in agreement with studies by Turk [28] showing that MTX at 12.5 mg/kg/day inhibited induction of CHS to oxazolone in the guinea pig, in keeping with similar results on delayed type hypersensitivity to tuberculin. Importantly, despite its well-documented anti-inflammatory effect in several models, MTX failed to decrease skin inflammation, edema, IFN-γ expression and CD8⁺ T cell accumulation when administered at the time of challenge. Finally complete inhibition of CHS induction was achieved at MTX doses that do not induce detectable alterations in thymus and peripheral lymphoid tissues [22]. These results suggest that the therapeutic effect of MTX in chronic dermatoses involving pathogenic mechanisms similar to those of CHS, may be attributed to its interference with clonal expansion but not to the inhibition of peripheral inflammatory events during the effector phase.
The prodrug MMF and its active metabolite MPA are primarily cytostatic agents that block activated T cells in the G1 phase of the cell cycle [29, 30]. The blocking effect of MMF on CHS induction is quite likely to be attributed to drug interference with clonal expansion, as is the case for MTX and 5-FU. However additional mechanisms could contribute to this effect, including interference of MMF with the final differentiation of dendritic cells. Indeed Mehling and coworkers [31] showed that MMF (150 mg/kg) injected at the time of induction or challenge moderately decreased (32 to 38%) CHS to oxazolone in BALB/c mice. Furthermore the differentiation of dendritic cells from bone marrow cells in vitro was impaired by addition of MMF. In the present study MMF did not interfere with the migration of Langerhans cells to the draining lymph node. Moreover the expression of high density of MHC class II molecules provided further evidence that dendritic cells had differentiated into fully competent antigen presenting cells. Therefore the contribution of dendritic cells to the suppression of CHS induction by MMF does not seem a major one. The data are consistent with the demonstration in other models that cell division is an absolute requirement for the differentiation of effector cytotoxic T cells [32].
The intercalating agent MXN was shown here to induce apoptosis of non activated as well as Con A-activated mouse lymph node cells (Fig. 1), in keeping with its cell-cycle independent toxicity to human T and B lymphocytes (Ferraro et al., manuscript in preparation). In this respect MXN is comparable to anthracyclins [33]. Although these drugs target DNA, they may also act on cells in the G0 phase of the cell cycle. Indeed we show here that a single injection of this drug is sufficient to induce a profound lymphoid depletion, involving B, CD4⁺ and CD8⁺ T lymphocytes without subset selectivity. If the drug was cell cycle dependent as are MTX, 5-FU and MMF, lymphocyte depletion would require a long period of administration because only a small percentage of lymphocytes are cycling in peripheral lymphoid organs. Inhibition of CHS induction by MXN most likely results from the massive CD8⁺ T cell depletion, although the additional contribution of other cell types (e.g. dendritic cells) can not be formally excluded. Most importantly, MXN injected at the time of DNFB challenge completely abrogated edema, CD8⁺ T cell infiltration and subsequent IFN-γ expression. This is quite remarkable in view of the very rapid kinetics of events following challenge. Chemotactic activity and C5a are detectable within 1-2 h after DNFB application and IFN-γ expression by CD8⁺ T cells at 4 h [34]. Therefore MXN must destroy or inactivate differentiated effector cytotoxic CD8⁺ T cells within a few hours after its injection.
CYP in vivo displays the same properties as MXN: a single injection is sufficient to induce B but also T cell depletion in peripheral lymphoid organs (Fig. 2). Furthermore CYP, like MXN, not only inhibits induction of CHS but also prevents its expression when injected intraperitoneally 4 h before the time of challenge. CYP was initially reported to selectively deplete B cells in spleen and lymph nodes following a single injection of 300 mg/kg in mice [35] then to act selectively on rapidly dividing cells, including activated T cells in the deep cortex during sensitization to haptens. Hence CYP administration did not fully prevent ³[H]-thymidine incorporation into lymph node cells in vivo but either blocked blastogenesis or induced the shrinkage of blast cells [28]. Although we have no in vitro data to demonstrate a toxicity of CYP metabolites towards non cycling T cells, our in vivo results clearly exclude a cell cycle dependent effect. Most reports on CYP were focused on the enhancement of CHS, Jones-Mote reaction, delayed hypersentivity to soluble proteins or to sheep red blood cells when CYP was administered prior to sensitization at low doses that do not induce lymphocyte depletion [16, 36]. It was then assumed that these reactions were normally down-regulated by a population of suppressor T cells exquisitely sensitive to low dose CYP. More recently P.W. Askenase and coworkers [17] reported that CYP (50 mg/kg) inactivated CD3⁺ CD8⁺ non antigen-specific natural suppressor T cells that prevented the expression of CHS in recipients of sensitized αβ T cells. Obviously quite distinct effects of different doses of CYP on CHS are to be anticipated. The protocol used in the present experimental study is very close to the clinical use of CYP which is routinely given as monthly infusions of 150 mg/kg in autoimmune diseases and vasculitis. We assume that the effects of MXN and CYP on CHS on the present experimental model are due to T cell depletion and may be extended to other “lymphoablative regimens” that share similar depleting effects.
Most studies on the pathophysiology of CHS have traditionally focused on the sensitization phase. However, for clinicians, the effector phase is much more important, because sensitization is generally asymptomatic, whereas elicitation of CHS results in manifestation of allergic contact dermatitis. We show here that three antimetabolites, including two currently used immunosuppressive agents MTX and MMF, inhibit CHS when administered during induction but not during the time of challenge. These drugs are cell cycle dependent and most likely interfere with clonal expansion of CD8⁺ effector T cells. Conversely, CYP, an alkylating drug and MXN, a DNA intercalating agent, not only prevent induction but also inhibit the expression of CHS when injected at the time of challenge as a single dose similar to those used in clinical applications. This activity correlates with a T and B lymphocyte depletion that involves non cycle dependent toxicity. n

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