MD41, a novel T helper 0 clone, mediates mast-cell dependent delayed-type hypersensitivity in mice

Abstract

In a previous study on mouse, we have shown that delayed-type hypersensitivity (DTH) could be classified into two types according to MC requirement. The first type of DTH could be elicited by sensitization with methylated human serum albumin (MHSA) in complete Freund's adjuvant (CFA) in both wild type and mast-cell deficient (W/W^v) mice. The second type could be elicited by MHSA in incomplete Freund's adjuvant (IFA) sensitization in wild type but not W/W^v mice. While the former was related to classic tuberculin (tbc)-type DTH, the latter appeared to be a novel mast-cell dependent DTH (MD-DTH). In order to investigate the mechanism of MD-DTH, in this study, we generated an effector T-cell clone (MD41) from lymph node cells of MHSA in IFA-sensitized mice and analysed its pattern of cytokine production. Our results from cytokine assays show that following antigen stimulation, MD41 cells produce significant amounts of the T helper 1 (Th1) cytokine interferon-γ (IFN-γ) as well as the Th2 cytokines interleukin (IL)-4 and IL-10. In addition, double staining for IL-4 and IFN-γ revealed that MD41 cells produce both Th1- and Th2-type cytokines simultaneously, which suggest that MD41 represents a Th0 clone rather than a mixture of Th1 and Th2 clones. Adoptive transfer of MD41 cells into wild-type mice resulted in the development of DTH skin reactions similar to those produced by active sensitization, with very similar histological findings. However, DTH skin reactions could not be induced in W/W^v mice unless first reconstituted with normal bone marrow MC (BM-MC). Therefore, our study suggests that in conjunction with tissue MC, MD41, a less-polarized MD-DTH-derived Th0 clone, is capable of developing murine DTH to the same extent as strongly polarized Th1 cells and mediates MD-DTH rather than tbc-type DTH.

Introduction

In vivo T-cell mediated immune responses, such as delayed-type hypersensitivity (DTH) and contact sensitivity (CS), depend on interactions between a variety of cell types that contribute to the final inflammatory response and tissue swelling. Local MC (MC) play a critical role in these immune responses with the release of the vasoactive amine serotonin (5-HT) that mediates early initiating events. Askenase et al.^1–3 reported that DTH in contact-sensitized mice is characterized by two sequential T-cell activities, an early skin swelling reaction peaking 2 hr after challenge, and a conventional delayed-type skin reaction peaking 24–48 hr after challenge. The authors then suggested that both these T-cell dependent inflammatory reactions require the involvement of MC.⁴ However, using the same experimental system, other investigators argued that MC-deficient mice (W/W^v), whose skin contained only 1% of normal MC number, could evoke DTH skin responses at almost the same level as that observed in normal control mice.^5–8 To resolve this apparent discrepancy, many investigators have focused their attention on the immunoregulatory potential of MC. Indeed, the requirement for MC in DTH may be dependent on the strength of the immune response, which is affected by various factors including allergen/hapten concentration, the adjuvant/solvent used for sensitization and response elicitation, and living conditions that may influence the activation status of memory cells and APCs.⁹ Thus, the detailed behaviour of MC during the establishment of DTH and the interactions between MC and other cell populations, such as lymphocytes or stromal cells, needs to be clarified.

While MC have been shown almost exclusively to be the critical effector cell type in immunoglobulin E (IgE)-mediated allergic reactions, reports have also indicated that MC may have other important roles in both specific and innate immune responses.¹⁰ Following high-affinity IgE receptor (FcεRI)-mediated activation, or other stimuli, MC undergo degranulation that results in the release of histamine, proteoglycans, neutral proteases, and lipid-derived mediators. At the same time, MC also release multifunctional cytokines and chemokines that influence physiological, immunological and pathological processes.¹¹ With regard to the relationship between MC and DTH, Askenase et al.¹² proposed a new paradigm indicating that MC are stimulated through complement receptors that recognize antigen bound to IgM antibodies, produced through natural immunity. MC then release 5-HT, tumour necrosis factor-α (TNF-α), and interleukin (IL)-1β that initiate hyperpermeability and promote the migration of antigen-specific T cells and antigen-presenting cells (APCs). Thus, the profound involvement of MC in this model strongly supports the concept that early DTH events, perhaps directly mediated by 5-HT release, even in the absence of detectable early macroscopic swelling at the site of antigen challenge, may initiate subsequent T-cell mediated immune responses in vitro.¹³ Additionally, some investigators have reported that T-cell factors, such as histamine-releasing factors,^14–16 IL-3,^16,17 and interferon-γ (IFN-γ)^16,17 affect MC and stimulate the release of chemical mediators that evoke local inflammation. Recently, Biedermann et al.¹⁸ reported that tissue MC are not only involved in the initiation and amplification of DTH, but also determine the pattern of inflammation through the type of chemokines and cytokines produced.

Upon antigen stimulation, naive CD4⁺ T cells differentiate into distinct effector cell populations based on cytokine secretion patterns and immune regulatory functions.^19,20 T helper 1 (Th1) cells primarily secrete IL-2, IFN-γ, TNF-α and TNF-β, while Th2 cells secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13.^21,22 Th1 cells tend to be involved in cell-mediated immunity, such as DTH and macrophage activation, while Th2 cells tend to be involved in humoral immune responses primarily by enhancing the production of selected antibody isotypes and eosinophils proliferation and function.^23–26 However, Some T-cell clones have been identified as having the capacity to produce both Th1 and Th2 cytokines, and are termed Th0. These cells are thought to represent a stage of differentiation before commitment to the Th1 or Th2 lineage.^21,22,27

In a previous report, we demonstrated that the DTH response to methylated human serum albumin (MHSA) emulsified in complete Freund's adjuvant (CFA) could be evoked in MC-deficient mice (W/W^v) to a similar or slightly lesser degree than in normal littermates.²⁸ At the same time, using a different sensitizing protocol in which MHSA was emulsified in incomplete Freund's adjuvant (IFA), we also described a novel type of DTH that developed in wild-type mice but not in W/W^v mice. Based on the requirement for MC in DTH responses and the sensitization protocol, we speculated that DTH in mice could be subdivided into two types, one probably represented by the classic tuberculin (tbc)-type DTH, and the other a novel MC-dependent DTH (MD-DTH). In addition, while the tbc-type DTH skin reaction could be suppressed by inhibition of macrophage function, MD-DTH was not affected by the same pretreatment, which suggested that MC rather than macrophages played a pivotal role in MD-DTH.²⁹ However, characterization of the effector T cells contributing to MD-DTH remained unresolved.

In the present study, we examined the cytokine secretion pattern of a T-cell clone, MD41, established from the lymph node cells of MHSA- and IFA-sensitized mice and determined whether DTH skin reaction could be elicited by the adoptive transfer of MD41 cells.

Materials and methods

Mice

C57BL/6 SlcCr mice, BALB/c mice, MC-deficient WBB6F1-W/W^v (W/W^v) mice, and normal WBB6F1+/+littermates, were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and bred at the Animal Institute of Shimane Medical University (Izumo, Japan). Age- and sex-matched mice 3–4 months old were used in all experiments.

Antigen and sensitization

Crystallized human serum albumin (HSA) was purchased from UCB-Bioproducts (Brussels, Belgium) and crystallized ovalbumin (OVA) was purchased from Nutritional Biochemical Co. (Cleveland, OH). Methylated HSA (MHSA) and methylated ovalbumin (MOA) were prepared by methylating the carboxyl groups using the methanol-hydrochloric acid method of Crowle et al.³⁰ Sensitization was performed as previously described.^31,32 Briefly, 5 mg/ml MHSA was emulsified with an equal volume of IFA, and 0·05 ml of the emulsion was injected s.c. into both hind footpads of mice.

Monoclonal antibodies and reagents

Hybridomas G.K.1 5 (anti-mouse CD4, rat IgG2b) and 11b11 (anti-mouse IL-4, rat IgG1) were obtained from the American Type Culture Collection (Rockville, MD). Biotin-labelled rat anti-mouse CD90.2 (Thy1.2, 53-2.1, rat IgG2a), biotin-labelled rat anti-mouse CD8 (Ly-2, 53-6.7, rat IgG2a), biotin-labelled rat anti-mouse CD45R (B220, RA3-6B2, rat IgG2a), fluoroscein isothiocyanate (FITC)-conjugated anti-rat immunoglobulin, and phycoerythrin (FE)-conjugated anti-IFN-γ (XMG1.2, rat IgG1) were purchased from Pharmingen Inc. (San Diego, CA). Brefeldin A and saponin were purchased from Sigma Chemical Co. (St. Louis, MO). Concanavalin A (con A) was purchased from Honen Cooperation (Tokyo, Japan). Mitomycin C (MMC) was purchased from Kyowa Hakko Kogyo Co. (Tokyo, Japan).

Establishment of the T cell clone (MD41)

Four weeks after sensitization with MHSA in IFA, mice were killed and their drained lymph node cells were dissected and suspended in Iscove's modified medium containing 10% fetal calf serum (FCS). For primary in vitro antigen stimulation, pooled cells (2·3 × 10⁷) from four lymph nodes of two MHSA in IFA sensitized mice were used. Lymph node cells (5 × 10⁶) and MMC-treated erythrocyte-depleted syngeneic spleen cells (1 × 10⁷), used as antigen-presenting cells (APC), were cocultured in the presence of 10 or 20 µg/ml MHSA in 5 ml medium in a tissue culture dish (Becton Dickinson, Franklin Lakes, NJ). After 4 days, the activated cells were harvested and subjected to primary cloning. Briefly, 10 responder cells and 4 × 10⁵ MMC-treated stimulator cells were seeded into four microplates with 96 round-bottomed wells each (Intermed, Roskilde, Denmark) in the presence of 5 µg/ml MHSA in 200 µl Iscove's modified medium supplemented with 20% con A-stimulated rat spleen cell culture supernatant (con A-sup). In all cases, Iscove's modified medium supplemented with 20% con A-sup was used for expansion of sensitized T cells. Half the culture medium in each well was replaced with fresh conditioned medium at 3- to 4- day intervals until colony formation was identified by phase contrast microscopy. Out of the 384 wells used, 87 were able to support cell growth. Cells, selected from these 87 wells, were examined for antigen-specific response by isotope (³H-TdR) incorporation about 20 days later. Five colonies in the 87 tested wells showed significant levels (approximately 1–6 × 10³ disintegrations per minute (d.p.m.) of isotope incorporation, and were continuously maintained and carefully expanded in the medium as described above. Following screening by antigen-specific ³H-TdR incorporation and flow cytometrical analysis, two cell lines, which consisted of CD4 positive helper T cells, survived and one of them was re-cloned using the limiting dilution method. After 2 weeks culture with half-medium changes, three colonies from 96 wells were expanded by antigen stimulation, and only one clone (MD41) was established as a stable growing line. Cells derived from the other two clones could not be analysed because of insufficient growth and incomplete characterization. On the other hand, cells derived from the MD41 clone showed stable proliferation under both regular antigen stimulation and resting culture conditions, although over 3 months were needed to establish a stable and useful T-cell clone. MD41 cells were frozen at −80° until use. For all experiments, resting MD41 cells were subcultured for 2 weeks following antigen stimulation, with restimulation and expansion at 2-week intervals.

Proliferation assay

MD41 antigen specificity and restriction to major histocompatibility complex (MHC) were examined as follows. MD41 cells (1 × 10⁵) were cultured with 5 × 10³ irradiated spleen cells in 96-well microplates in a total volume of 0·2 ml Iscove's modified medium supplemented with 0·3% normal mouse serum with or without protein antigen for 2 days, with 1 µCi ³H-TdR added for the last 12 hr of culture. Cells were harvested onto glass fibre filters and the incorporated radioactivity was determined by scintillation counting. All cultures were performed in triplicate.

Flow cytometric analysis

For the analysis of MD41 surface antigens, cells suspended in Hank's balanced salt solution (HBSS) supplemented with 0·1% NaN₃ (HBSS/NaN₃), were incubated with optimally diluted anti-CD4, biotin-labelled anti-CD90.2, anti-CD8 or anti-CD45 monoclonal antibodies on ice for 30 min. After two washes with HBSS/NaN₃, the cells were incubated with FITC-labelled rat immunoglobulin or FITC-labelled avidin on ice for 25 min. Finally, after another two washes with HBSS/NaN₃, the cells were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). For the double staining of intracellular cytokines, MD41 cells were resuspended at 10⁵−10⁶/ml in Iscove's modified medium supplemented with 10% FCS and stimulated with con A at 5 µg/ml or phorbol 12-myristate 13-acetate (PMA) at 10 ng/ml for 48 hr. Brefeldin A at 10 µg/ml from a stock solution of 1 mg/ml in dimethyl sulphoxide (DMSO) was then added, and after 2-hr incubation at 37°, the cells were harvested, washed, and fixed in an equal volume of 4% paraformaldehyde. After fixing for 20 min at room temperature, the cells were stained immediately for cytokines determination. For cytokines intracellular staining, all reagents and washes contained 1% bovine serum albumin (BSA) and 0·5% saponin (HBSS/BSA/saponin), with all incubations performed at room temperature. The cells were washed with HBSS/BSA/saponin, preincubated for 10 min in HBSS/BSA/saponin, and then incubated with anti-IL4 (11B11) at 5 µg/ml or isotype-matched control (isotype control) at 10 µg/ml for 30 min. After two washes with HBSS/BSA/saponin, optimal concentrations of FITC anti-rat immunoglobulin (Pharmingen) were added for 30 min. After further two washes with HBSS/BSA/saponin, purified rat immunoglobulin was added at 300 µg/ml for 10 min to block residual anti-rat immunoglobulin binding. Without washing, phycoerythrin (PE)-conjugated anti-IFN-γ (XMG1.2) or control PE-conjugated rat immunoglobulin (Pharmingen) was added to a final concentration of 5 µg/ml. After 20 min, cells were washed twice with HBSS/BSA/saponin and then with HBSS/BSA without saponin to allow membrane closure. All samples were analysed on a FACScan flow cytometer (Becton Dickinson & Co.).

Adoptive transfer of MD41

MD41cells in the resting state were harvested and twice washed with minimal essential medium (MEM). For systemic transfer of MD-DTH, normal naive recipients mice were intravenously injected through the retro-ocular venous plexus with 1–5 × 10⁶ of MD41 cells in 200 µl PBS. After an interval of a few hours, recipient mice were intradermally challenged with 20 µg MHSA or unrelated protein antigen in the right footpad. Control mice were challenged with PBS only. For local transfer, 1–5 × 10⁶ of MD41 cells suspended in MHSA solution or unrelated antigen solution were intradermally injected to the right footpad of normal recipient mice. Footpad reactions were measured as the difference in thickness between challenged and unchallenged footpads under a dissecting microscope after 24 hr, and expressed in 1/10 mm. Antigen-challenged footpad skins were removed at 3 or 24 hr after challenge injection and fixed with 10% formaldehyde for routine haematoxylin and eosin staining. All groups consisted of four to five mice.

Cytokine production by mitogens or antigen-stimulated MD41 cells

For mitogen stimulation, supernatants of MD41 cultures stimulated with optimal concentrations of con A or MHSA were collected 48 hr after stimulation. For antigen stimulation, supernatants were collected 48 hr after coculture of MD41 cells, MMC treated spleen cells as APC and/or 20 µg MHSA. The amounts of cytokines IFN-γ, IL-4, and IL-10 produced in the culture supernatants were measured using ELISA kits (BioSource International Inc, Camarillo, CA).

Mast cell preparation and reconstitution

Bone marrow-derived connective tissue-type MC were obtained using the method of Kitamura et al.,³³ and 1 × 10⁶ matured MC were intradermally injected into the footpad 2 weeks prior to antigen challenge.

Results

MD41 characterization

In order to elucidate the mechanism of MD-DTH, MD41 cells were generated as described in Materials and Methods. Characterization of these MD41 cells indicated that T-cell markers such as CD90.2 (95%) and CD4 (99%) were strongly expressed in almost all MD41 cells, whereas suppressor or cytotoxic T-cell markers such as CD8, or CD45R were not detectable (Fig. 1).

Figure 1.

Phenotypic characterization of the MD41 clone established from lymph node cells of mice sensitized with MHSA in IFA. MD41 cells were stained with monoclonal antibodies against the lymphocyte markers CD90·2 (a), CD4 (b), CD8 (c), and CD45R (d). After two washes, the cells were analysed by flow cytometry. Fluorescence intensity of background staining (…) or specific binding in the presence of primary mAbs (—) was determined using a FACScan.

In order to examine the response of MD41 cells to specific antigen and MHC class II restriction, proliferation in response to the sensitizing protein MHSA and other unrelated proteins such as HSA and MOA was measured using the isotope incorporation assay. As shown in Fig. 2, compared with stimulation by unrelated antigens, which led to similar levels of proliferation as controls, MD41 cells showed significantly higher levels of ³H-TdR incorporation in response to MHSA in a dose-dependent manner. For MHC restriction analysis, MMC-treated spleen cells from mice with a different MHC class II-haplotype were used as the APC. A significant response by MD41 cells was observed when haplotype-matched C57BL/6 splenocytes were used as APC but not when haplotype mismatched splenocytes were used (Table 1). These results demonstrated that MD41 is an antigen-specific, MHC-restricted helper T-cell clone.

Figure 2.

MD41 cells show a proliferative response to the sensitizing antigen. Resting MD41 cells (1 × 10⁵) were cultured with mitomycin c-treated syngeneic spleen cells (5 × 10³) used as APCs in the presence of either MHSA (open columns), HSA (hatched columns), or MOA (closed columns) in 96-well microplates. Cells were cultured for 2 days with 1 µCi ³H-TdR added to each well for the last 12 hr of culture. ³H-TdR incorporation was determined using a liquid scintillation counter. Each value represents an average of five or six mice ± SE.

Table 1. Restriction of MD41 to MHC class II molecules.

Responder MD41 (1 × 104 cells)	Stimulator mouse strain	MHC class II haplotype	Antigen (20 μg/ml)	3H-TdR incorporation (× 10−4 DPM)
+	AKR	k	MHSA	0.4
+	BALB/C	d	MHSA	0.4
+	C3H/He	k	MHSA	0.5
+	DBA/2	d	MHSA	0.4
+	NZB	d	MHSA	0.5
+	NZW	z	MHSA	0.5
+	C58BL/6	b	MHSA	7.0
+	C57BL/6	b	MOA	0.4
	C57BL/6	b	MHSA	0.4

Adoptive transfer of MD41 cells in naive mice

As the development of DTH skin reactions is mediated by circulating sensitized CD4⁺ T cells, we examined whether MD41 cells had the capacity to elicit DTH skin reactions in naive mice. The magnitude of delayed footpad swelling mediated by MD41 transfer is shown in Table 2. After transfer of MD41 cells, significant footpad swelling was induced in naive C57BL/6 mice regardless of the route of cells transfer, with responses showing only slight variations in magnitude. In contrast, MHSA-induced reactions were undetectable in mice not given MD41 cells (data not shown). Also, delayed footpad swelling was undetectable when recipient mice were challenged with unrelated antigen (MOA), or when MD41 cells were transferred to allo-recipient mice (BALB/c), which suggest that MD41-mediated footpad responses were developed in an antigen-specific and MHC-restricted manner. In contrast, footpad swelling after intravenous transfer of MD41 cells into W/W^v mice was almost undetectable despite the transfer of 10 times as many cells as transferred in normal mice. Footpad swelling was elicited in W/W^v mice by the transfer of bulk lymph node cells from MHSA and CFA-sensitized mice but not by those from MHSA and IFA sensitized mice. Therefore, our results indicate that MD41 cells have the capacity to elicit DTH skin reactions in C57BL/6 but not in W/W^v mice.

Table 2. Footpad reaction by systemic and local adoptive transfer of MD41 cells.

	Cell transfer
Source	Dose/mouse	Route§	Recipient	Challenge	FPR at 24 hrs (mean ±S.E.)¶
MD41*	1 × 106	s.c.	C57BL/6	MHSA	0.6 ± 0.2
	5 × 106	s.c.	C57BL/6	MHSA	5.0 ± 0.5
	5 × 106	s.c.	C57BL/6	−Å	0
	1 × 106	i.v.	C57BL/6	MHSA	1.3 ± 0.3
	5 × 106	i.v.	C57BL/6	MHSA	1.5 ± 0.4
	1 × 106	i.v.	C57BL/6	MOA	0
	1 × 106	i.v.	C57BL/6	−Å	0
	5 × 106	i.v.	BABL/c	MHSA	0
	1 × 106	i.v.	W/Wv	MHSA	0
MHSA and CFA †sensitized T cells
	4 × 106	i.v.	C57BL/6	MHSA	7.2 ± 1.3
	4 × 106	i.v.	W/Wv	MHSA	3.8 ± 0.4
MHSA and IFA ‡sensitized T cells
	4 × 106	i.v.	C57BL/6	MHSA	1.2 ± 0.4
	4 × 106	i.v.	W/Wv	MHSA	0

^*MD41 suspended in PBS were injected into recipients via the s.c. or i.v. route. s.c., subcutaneous injection; i.v., intravenous injection.

^†Nylon wool passed lymph node cell immunized with MHSA and CFA in both footpads and 8–12 days before were for the transrfer.

^‡Nylon wool passed lymph node cell immunized with MHSA and IFA in both footpads and 4 weeks before were for the transrfer.

^§For the systemic trasnfer recipients received with MD41 or sensitized T cell through i.v. route were challenged with antigen at right footpad. For the local transfer, recipients were subcutaneously injected with MD41 in antigen solution. After 24 hrs, footpad reaction (FPR) of right footpad was measured as described in Materials and Methods.

^¶Each value represents an average orf four or five mice ± S.E.

To further characterize the DTH response induced by MD41 cells' adoptive transfer, we performed histological examinations of the MHSA-challenged sites. In C57BL/6 mice that received i.v. injection of MD41 cells, a large number of mononuclear cells, including neutrophils, lymphocytes, and macrophages, were found to have accumulated at the site of antigen challenge 24 hr after MD41 administration (Fig. 3c, d). The lesions were more exudative and the infiltration of neutrophils was more predominant than observed for tbc-type DTH (Fig. 3d). While the mononuclear cells appeared to be a mixture of lymphocytes, MD41 cells, macrophages, stromal cells, and MC, definitive identification was not performed in this study. In contrast to the histological changes observed 24 hr after MD41 administration, few inflammatory cell infiltrates were observed in the lesions 3 hr after challenge injection in C57BL/6 mice (Figs 3a,b). However, a small but significant number of MC demonstrating degranulation were focally detected (Fig. 3b, arrowed). There was no evidence of direct interaction between MC and other mononuclear cells. Control C57BL/6 mice that received adoptive transfer of MD41 cells but not the antigen challenge were almost devoid of inflammatory cells infiltration (Fig. 3e). In the case of BM MC-reconstituted W/W^v mice (Figs 3f,g), pathological findings at 24 h after challenge injection were very similar to those of C57BL/6 mice. Furthermore, no marked infiltration by inflammatory cells was observed in unreconstituted W/W^v mice that received adoptive transfer of MD41 cells (Fig. 3h). These histological results indicate that MD41 cells are able to induce DTH skin reactions in MC sufficient mice, but not in MC deficient W/W^v mice. Additionally, DTH skin reactions by MD41 could be restored in W/W^v mice by reconstitution with BM MC suggesting that footpad response induced by MD41 might correspond to MD-DTH.

Figure 3.

MD41 cells develop DTH skin responses in footpads of C57BL/6 but not W/W^v mice. C57BL/6 mice were intravenously injected with 2 × 10⁷ MD41cells (a–d). After an interval of a few hours, recipient mice were intradermally challenged with 20 µg MHSA in the right footpad. As a control, mice were intravenously injected with MD41 but not challenged with MHSA (e). Challenged footpad skins were removed at 3 hr (a, b) or 24 hr (c, d, e) after injection and processed for the histological examination. W/W^v mice reconstituted with 1 × 10⁶ BM MC 2 weeks earlier in the right footpad (f, g) or not (h) were intravenously injected with 2 × 10⁷ MD41 and challenged with MHSA in the same way as C57BL/6 mice. Massive infiltration of mononuclear cells including neutrophils (▴), lymphocytes, and macrophages were observed 24 h after challenge in C57BL/6 (c, d) and BM MC reconstituted W/W^v (f, g) mice. The proportion of neutrophils in both groups was higher than that of tbc-type DTH. While only mild haemorrhaging and oedema without massive cell infiltration was observed in early skin lesions (a, b), degranulation of MC was focally obvious (square inset in a is magnified in b). No marked DTH skin reactions were observed in W/W^v mice unreconstituted with BM MC (h).

Cytokine production pattern of MD41 cells

In an attempt to determine the pattern of cytokines produced by MD41, we measured the amounts of cytokines secreted into the culture supernatants of antigen- or mitogen-stimulated MD41 cells. As shown in Fig. 4, when stimulated with MHSA, MD41 cells produced large amounts of IL-4, and small but significant amounts of IL-10 and IL-3 (IL-3; data not shown). At the same time, MD41 cells were also capable of producing Th1-type cytokine IFN-γ under the same stimulation. However, MD41 cells did not produce detectable amounts of these cytokines in the absence of MHSA stimulation (data not shown). The pattern of cytokine production after mitogen stimulation using Con A (Fig. 4) or PMA (data not shown) was similar to that of MHSA stimulation, and significant amounts of both types of cytokines were produced. These results suggest that MD41 cells have the capacity to secrete both Th1- and Th2-type cytokines after antigen as well as mitogen stimulation.

Figure 4.

MD41 cells secrete Th1- and Th2-type cytokines after antigen or mitogen stimulation. Resting MD41 cells were stimulated with syngeneic mitomycin c-treated spleen cells in the presence of MHSA (20 µg/ml) or with Con A (5 µg/ml). Supernatants were collected at 48 hr and cytokine ELISAs for IFN-γ (open columns), IL-4 (hatched columns), and IL-10 (dotted columns) performed (as described in Materials and Methods). Vertical bars represent SE.

However, analysis of cytokine production from culture supernatants gave no information on the individual cell within the population, or the level of heterogeneity or commitment to cytokine production at single cell level. To confirm that MD41 cells consisted of only Th0 cells, we performed intracellular cytokine staining. As shown in Fig. 5 and according to single-staining flow cytometry, the majority of cells stimulated with con A for 48 hr produced IFN-γ (a: 83·4%) or IL-4 (b: 74·4%). At the same time, double staining with antibodies against these cytokines demonstrated that a significant proportion of MD41 cells produced IFN-γ and IL-4 simultaneously (c: 81%) and that single positive populations were rare (IFN-γ: 3%, IL-4: 5%). Further analysis by reverse transcriptase–polymerase chain reaction showed that mRNA encoding IL-4, IL-10, type 2 and IFN-γ, type1, cytokines were expressed in antigen-stimulated MD41 cells (data not shown). Thus, these results strongly support the idea that MD41 represented a Th0 clone rather than a mixture of Th1 and Th2 clones.

Figure 5.

MD41 cells simultaneously produce IL-4 and IFN-γ at single cell level after the Con A stimulation. MD41 cells cultured with Con A or medium alone for 24 hr were fixed in 4% paraformaldehyde and washed with HBSS supplemented with 0·5% saponin. Cells were stained with rat anti IL-4 and FITC-labelled anti-rat immunoglobulin as described in Materials and Methods. After two washes with HBSS containing 0·5% saponin, cells were stained with PE conjugated anti-IFN-γ. After 20 min, cells were washed twice with PBS/BSA/saponin and then with PBS/BSA without saponin to allow membrane closure. Samples were analysed on a FACScan flow cytometer. Frequencies of IFN-γ-producing cells are shown on the y-axis and IL-4-producing cells on the x-axis. Staining with anti-IFN-γ-PE alone (a), staining with anti-IL-4 alone (b), and double staining (c).

Discussion

In the present study, we demonstrated that a T-cell clone with a Th0-type cytokine production pattern could be generated from drained lymph node cells of MHSA in IFA-sensitized mice. We also showed that DTH skin reactions could be elicited in naive C57BL/6, but not in W/W^v mice by adoptive transfer of MD41 cells and that these reactions were similar to those observed in active sensitization of MD-DTH. In addition, the defective MD-DTH exhibited by W/W^v mice was restored after reconstitution with BM MC. These results strongly support our previous hypothesis indicating that two types of DTH exist in mice, one a MC-independent classic tbc-type DTH, and the other a MC-dependent MD-DTH.

It is very important to determine whether MD41 cells are a mixture of Th1 and Th2 clones or just a single clone. In order to obtain a cloned T-cell line, we used the limiting dilution method and re-cloned cells that grew under rough primary cloning conditions. Additionally, almost all MD41 cells established in this study (over 80%) produced both IL-4 and IFN-γ simultaneously (Fig. 4). While these results indicate the possibility that MD41 is a single clone, it is necessary to demonstrate single DNA arrangements in MD41 using DNA sequencing methods.

As described in our previous report,²⁸ kinetic studies on local skin inflammation have demonstrated that maximum footpad swelling in MD-DTH is evident about 4 weeks after MHSA in IFA sensitization, whereas tbc-type DTH shows maximum response 8–12 days after sensitization. This maximum response then subsides before 4 weeks from sensitization. Accordingly, in our preparation of MD41 cells, we have restricted the lymph node cells used to those taken 4 weeks after MHSA in IFA sensitization, and prevented the expression of tbc-type specific T cells in the primary culture system.

It is now clear that many murine and human T-cell clones do not fit the strict Th1/Th2 classification.³⁴ Clones derived from mitogen- or alloantigen-stimulated cultures, and/or assayed early after derivation, frequently coexpress Th1 and Th2 cytokines in various combinations. Such cells have been classified as a third subset and are noted Th0. However, the discrete classification of Th clones based on the definite amounts of cytokines secretion remains to be settled. Dittle et al. previously analysed the cytokine production of Th1, Th2 and Th0 clones bearing the same T-cell receptor and showed undetectable levels of IL-4 production by Th1 and IFN-γ production by Th2, respectively.³⁵ Furthermore, Orly et al. proposed two types of Th0 cells; Th0A cells that secrete IL-2 and IL-4, but not IFN-γ and Th0 B cells that secrete IL-2, IL-4, and IFN-γ. While the authors did not directly compare the amounts of IL-4 and IFN-γ produced by Th0 B cells, the level of IFN-γ produced by Th0 B cells was considerably less than that of IL-4.³⁶ In the present study, while mitogen or antigen stimulation induced excess production of IL-4, IFN-γ secretion was significantly remarkable with ratios of 3·9 : 1 (IL-4 : IFN-γ) and 1·9 : 1 (IL-10 : IFN-γ) after mitogen stimulation, compared to ratios of 3·4 : 1 (IL-4 : IFN-γ) and 1·7 : 1 (IL-10 : IFN-γ) after antigen stimulation. Thus, we conclude that MD41 can be classified as a Th0 clone.

Following the first report by Mosmann et al. the fact that mouse DTH is mediated by Th1 clones has been generally accepted.²³ From our current study, we propose the hypothesis that stimulation of local MC by MD41 demonstrating Th0 cytokines production pattern seems to be crucial for the development of DTH, particularly at early phases of inflammation. MC-derived factors, including vasoactive amines, cytokines and chemokines, together with other MD41-derived cytokines, may drive the subsequent response through activation of inflammatory DTH effector populations, as seen after 24–48 hr. Indeed, while we have not yet characterized MD41-derived factors, our preliminary data showed marked degranulation of MC cultured with MD41 and antigen under a separated culture system (unpublished data). As demonstrated in this study, MC degranulation in the challenged site was observed 3 hr after injection, suggesting a pivotal role of MD41 on MC degranulation (Fig. 5). As described in our previous report, MC, rather than macrophages appear to play central roles as inflammatory effector cells in the initiation and progression of the MD-DTH response.^28,29 From these in vivo and in vitro findings, degranulation of MC by MD41 profoundly contribute to the development of DTH known as MD-DTH, by contrast, MD41 cells are not able to induce significant levels of tbc-type DTH probably due to their low production of IFN-γ. It can therefore be speculated that the MD41 clone is more specific to MD-DTH than to tbc-type DTH and that a cytokines balance, in which degranulation of MC is predominantly preceded, strongly affects the determination and modification of DTH pattern. Askenase and coworkers suggested that sensitized T cells were capable of releasing factors possessing bidirectional binding activity to both antigens and putative receptors on MC.¹⁴ Factor-stimulated MC would then induce further inflammatory responses, such as increased vascular permeability with local edema, and the recruitment of leucocytes, finally producing skin lesions characteristic of late-phase DTH reactions.^15–17 Further studies on the functions of MD41 cells including their interactions with MC and specific effects on MD-DTH are needed.

In conclusion, this study has shed light on the differences between tbc-type DTH and MD-DTH. Particularly, we expect that MD41 cells will provide much information to clarify the mechanisms involved in DTH and help assess the responses mediated by other Th clones in DTH.

Abbreviations

DTH

delayed-type hypersensitivity

CS

contact sensitivity

HAS

human serum albumin

MHSA

methylated human serum albumin

APCs

antigen presenting cells

MOA

methylated ovalbumin

CFA

complete Freund's adjuvant

IFA

incomplete Freund's adjuvant

MC

mast cells

BM MC

bone marrow derived MC