Negative regulatory effect of histamine in DNFB-induced contact hypersensitivity

Edina Garaczi1, Márta Széll2, Tamás Jánossy3, Andrea Koreck1, Andor Pivarcsi1,2, Edit Buzás4, Zoltán Pos4, András Falus4, Attila Dobozy1,2 and Lajos Kemény1,2

1 Department of Dermatology and Allergology, 2 Dermatological Research Group of the Hungarian Academy of Sciences at the Department of Dermatology and Allergology, 3 Institute of Surgical Research, University of Szeged and 4 Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary

Correspondence to: E. Garaczi; Email: egaraczi{at}yahoo.com


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Histamine plays an important role in the regulation of various immunological functions. To evaluate the role of histamine in contact hypersensitivity, contact dermatitis was induced with dinitrofluorobenzene (DNFB) in histidine decarboxylase knockout (HDC–/–) histamine-deficient and wild-type mice. The DNFB-induced increase of the ear thickness was significantly higher in HDC–/– mice than in wild-type mice. Using flow cytometry, significantly lower percentages of CD4+ Th and CD8+ Tc cells, and significantly higher percentages of CD45R+ B cells were observed in the regional lymph nodes in HDC–/– mice than in wild-type mice. In the ear specimens of both groups, the majority of the infiltrating cells were neutrophils and macrophages at 24 and 48 h after challenge. Using immunohistochemistry, we observed significantly more CD45+ leukocytes in HDC–/– mice than in wild-type mice. The expression of Th1 (IL-2, IFN-{gamma}, TNF-{alpha}) and Th2 (IL-4) mRNAs was examined by quantitative real time RT–PCR in the ear samples. The levels of Th1 cytokine mRNAs both at 24 and 48 h after challenge and IL-4 mRNA at 48 h showed a significantly higher increase in HDC–/– mice than in wild-type mice. These results suggest that histamine plays a negative immunoregulatory role in DNFB-induced contact hypersensitivity.

Keywords: delayed type hypersensitivity, histidine decarboxylase, immunoregulation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Histamine is present in all tissues of the mammalian body and plays an important role in many physiological and pathological functions. The importance of histamine has been demonstrated in gastric acid secretion, contraction of smooth muscle, neurotransmission, wound repair, embryogenesis, hematopoiesis, allergic skin reaction and malignant growth. Histamine, synthesized by histidine decarboxylase (HDC), is produced mainly in mast cells, basophils and histaminergic neurons, but macrophages, dendritic cells and T lymphocytes also synthesize histamine (13). The production and release of histamine are modulated by various cytokines such as IL-1, IL-3, IL-5 and IL-8 (4). Histamine plays a regulatory role in Th1/Th2 balance at multiple points; however, the majority of histamine actions seems to promote Th2 responses (56). Four different membrane receptors of histamine (H1R, H2R, H3R and H4R) have been pharmacologically and molecularly characterized. One or more of these receptor types are expressed on many different cell types, including T cells, B cells, monocytes and neutrophils (5,7). The secretion of IL-2 and IFN-{gamma} from Th1 cells can be either inhibited or stimulated by histamine, and both effects are mediated via H2R receptors (4). It has been recently published that H1R is overexpressed on Th1 cells, while H2R is overexpressed on Th2 cells. H1R-deficient mice demonstrate suppression of Th1 cytokines and dominant secretion of Th2 cytokines. H2R-deficient mice show a significant enhancement of both Th1- and Th2-type cytokine secretion (8).

The contact hypersensitivity (CHS) response develops in two distinct phases: sensitization and elicitation. In the sensitization phase, mice exposed to contact allergen showed an increase in the percentage of antigen (Ag) specific Thy1+/CD5+/CD3/TCR/B220+ cells in the skin-draining lymph nodes (DLN) (9,10). These B220+ (CD45R) B cells produce IgM/IgG type antibodies that pass into the circulation and the extravascular tissues. These antibodies bind to receptors on the surface of mast cells and platelets and play a role in the increase of vascular permeability. Cytokines produced by Tc1 cells (IFN-{gamma}), Th1 cells (IL-2, IFN-{gamma} and TNF-{alpha}), Th2 cells (IL-4 and IL-10) and Langerhans cells (IL-12 and IL-18) are important for the optimal induction and initiation of CHS in DLN (1113).

The elicitation phase is characterized by two distinct phases. In the early phase of elicitation, the antigen bound by IgM/IgG type antibodies produced by B220+ B cells leads to mast cell and platelet activation. Release of serotonin and TNF-{alpha} from these cells results in an increased vascular permeability (1416). Geba et al. (17) found that delayed type hypersensitivity reaction (DTH) was either intact or only partially decreased in mast-cell deficient mice, and severe depletion of platelets with anti-platelet antibody strongly inhibited the contact hypersensitivity, especially in mast-cell deficient mice (18). These data suggest that serotonin and TNF-{alpha} are important mediators in the early phase of DTH.

In the later phase of elicitation (48–72 h after challenge), antigen-specific T cells ({alpha}ßT cells) are activated, resulting in the production of various cytokines. It is known that in the CHS reaction the main effector cells are IFN-{gamma} producing CD8+ Tc1 cells (1921). The CHS responses are also regulated by IL-2, IFN-{gamma} and TNF-{alpha}-producing CD4+ Th1 cells, and IL-4 and IL-10-producing CD4+ Th2 cells (12,20,2225).

Belsito et al. (26) reported that the H2R antagonist cimetidine augmented CHS reaction by inhibition of the induction of T-suppressor cells. In contrast to this, the histamine H1R antagonist diphenhydramine, had no effect on suppressor cell activity in the CHS reaction in mice (27), and H1R antagonists did not cause the downregulation of CHS. Grob et al. (28) tested the effect of a prolonged treatment with H1R antagonist cetirizine on the reaction to a contact allergen applied by patch testing in a sensitized population. Their results demonstrate that the clinical recording did not show any difference between the cetirizine treated and the control groups. These data suggest that histamine might be involved in the regulation of CHS through H2R receptor.

In the present study, we examined the CHS response in HDC knockout (HDC–/–) histamine-deficient mice. These mice were generated using a gene targeting method by Ohtsu et al. (29). HDC–/– mice exhibit a decreased number of mast cells. The lack of histamine leads to a large reduction in overall contents of mast cell secretory granules, including proteases MMCP4, MMCP5 (chymases) and MMCP6 (tryptase) (30). In HDC–/– mice plasma extravasation could not be observed after passive cutaneous anaphylaxis test (31), and histamine plays a significant role not only in the anaphylactic increase of vascular permeability but also in the negative regulation of neutrophil infiltration (6).

The purpose of the present study was to determine the immunoregulatory role of histamine in dinitrofluorobenzene (DNFB)-induced delayed type hypersensitivity. We found that the lack of histamine caused an intense Th1 type response, suggesting that histamine plays a negative regulatory role in contact dermatitis.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Generation of the HDC–/– mice was previously described (29). Female, 8–10-month-old HDC–/– and CD1 background wild-type mice were used in the experiments. Each experimental group consisted of 4–6 mice. The mice were kept on normal diet.

We previously published an impaired reproduction of histamine-deficient mice (32). Using CD1 background mice, the segregating F2 population contains a higher percentage of wild-type mice (>25%) than HDC–/– mice (<25%) (proportions are non-Mendelian) (unpublished data). Therefore, the HDC–/– mice were randomly selected from F2 mice of the transgenic colony.

Treatment
The abdominal skin of the mice was shaved and sensitized with 25 µl 0.5% 2,4-dinitrofluorobenzene (DNFB; Sigma-Aldrich Corporation, St Louis, MO) in acetone/olive oil (4:1) for two consecutive days (days 0 and 1). Five days later, the dorsal surface of both ears was challenged with 15 µl 0.2% DNFB. The control mice were also sensitized with DNFB, but their ears were treated with acetone/olive oil. Ear thickness was measured with a spring-loaded micrometer (Oditest; Dresden, Germany) before challenge and 24 and 48 h after challenge. Treated ears were harvested 24 and 48 h after the final application of DNFB or acetone/olive oil.

Flow cytometry
The axillary and inguinal lymph nodes draining the abdominal skin (sensitization area) were excised from each mouse 48 h following challenge. For phenotypic analysis by flow cytometry, individual cell suspensions were prepared in Dulbecco's phosphate-buffered saline (PBS) with 5% fetal calf serum (GIBCO BRL, Paisley, Scotland) and 0.1% sodium azide (Merck, Darmstadt, Germany) (PBS–FCS) at 4°C and washed by centrifugation at 350 g. The pellets were resuspended and diluted to 107 cells/ml in PBS with 1% bovine serum albumin (BSA, fraction V; Sigma-Aldrich Corporation, St Louis, MO) and 0.1% sodium azide (PBS–BSA). Cells were labeled with the following rat anti-mouse monoclonal antibodies: anti-CD45 (M1/89 clone), anti-CD3 (KT3 clone), anti-CD4 (H129.19 clone), anti-CD8 (53.6.72 clone), anti-CD45R (RA3-3A1 clone), anti-CD11b (M1/70 clone), anti-macrophage (F4/80 clone), anti-Gr-1 (RB6-8C5 clone). KT3, H129.19 and RB6-8C5 hybridomas were kindly provided by Professor W. Van Ewijk (Department of Immunology, Erasmus University, Rotterdam, The Netherlands), the other hybridomas were purchased from the American Tissue Type Collection (Rockville, MD). Tissue culture supernatants were produced by culturing hybridomas in RPMI 1640 with 2 mM L-glutamine, 10 mM Hepes, 2 g/l NaHCO3, 10% FCS (GIBCO BRL, Paisley, Scotland) and 5 x 10–5 M 2-mercaptoethanol (Sigma-Aldrich). Twenty-five microliters of cell suspensions was admixed to 25 µl samples of undiluted tissue culture supernatants in the wells of round-bottom microtiter plates. The plates were incubated at 4°C for 30 min, then the cells were washed three times with 200 µl/well PBS–FCS. The pellets were resuspended and incubated in 50 µl fluorescein isothiocyanate (FITC)-conjugated goat anti-rat Ig (Sigma-Aldrich) diluted to 1:200 at 4°C for 30 min. To avoid the cross-reactive binding of anti-rat Ig to mouse cell surface Ig, 2% normal mouse serum was admixed to the diluted anti-rat Ig. After washing, the cells were resuspended in 200 µl PBS–BSA and the dead cells were stained by adding 10 µl of 25 µg/ml propidium iodide (Sigma-Aldrich). 104 cells per sample were analysed with a FACStarPlus (with an argon ion laser, wavelength 488 nm; Becton Dickinson, Sunnyvale, CA). Data were analyzed and the percentages of positive cells were calculated with the Cell Quest 3.1F software (Becton Dickinson) (33).

Histology
Ear samples were taken 24 and 48 h after DNFB painting and fixed in 4 % formalin for routine histology with hematoxylin–eosin and toluidine blue staining. The sections were examined with objective 40x.

Immunohistochemistry
Fresh frozen skin specimens were embedded in cryomatrix (Life Sciences International, Shandon, UK), 3-µm serial cryostat sections were prepared and avidin–biotin–peroxidase complex (ABC) method was used for immunohistologic staining. The sections were air dried, acetone fixed, then incubated with 0.5% BSA (Sigma-Aldrich) before adding the primary antibodies (Pharmingen, Becton Dickinson Company): rat anti-mouse CD45 and rat anti-mouse CD3 monoclonal antibody. Normal rat serum (DAKO, Glostrup, Denmark) was used as negative control. The sections were incubated with biotin-conjugated rabbit anti-rat IgG (Vector Laboratories, Inc. Burlingame, CA), then with avidin–biotin–peroxidase (Vectastain Elite kit, Vector Laboratories). The peroxidase reaction was developed with 3-amino-9-ethylcarbazol (AEC; Sigma-Aldrich) and the sections were counterstained with hematoxylin.

Quantitative reverse transcription polymerase chain reaction (Q-RT–PCR)
Ear specimens taken 24 and 48 h after the DNFB treatment were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated following the instructions of the users' manual. RNA concentration was determined by the A260 value of the samples. First strand cDNA was synthesized from 3 µg total RNA in a 20 µl final volume by using a First Strand cDNA Synthesis Kit (MBI Fermentas, Vilnius, Lithuania). After reverse transcription, Q-RT–PCR was used to quantify the relative abundance of products of each gene (iCycler IQ Real Time PCR, Bio-Rad, Hercules, CA) using primers specific for mouse GAPDH, IL-2, IL-4, IFN-{gamma} and TNF-{alpha}. Two-microliter aliquots of the reverse transcription volume were used as templates for PCR reactions.

The sequences for primers specific for IL-2, IL-4, IFN-{gamma}, TNF-{alpha}, GAPDH are shown in Table 1. The conditions of the reactions were as follows: 95°C for 5 min followed by 40 cycles at 95°C for 15 s (denaturation) and at 57°C for 45 s (annealing and elongation). The Mg2+ concentration was 3 mM, the concentration of the primers was 300 mM. Real time detection of PCR products was carried out by using SYBR Green I dye.


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Table 1. The sequences of primer pairs used for Q-RT–PCR

 
Statistical analysis
Student's t-test was used for statistical evaluation; P < 0.05 was considered as significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
HDC–/– mice demonstrated increased contact hypersensitivity to DNFB
HDC–/– and wild-type mice were sensitized with 0.5% DNFB for two consecutive days. Five days later, the dorsal surface of the ears was challenged with 0.2% DNFB or with the solvent (acetone/olive oil). Ear thickness was measured before challenge, 24 and 48 h after challenge. Twenty-four hours after challenge, the DNFB-induced increase of the ear thickness was significantly higher in the HDC–/– mice than in wild-type mice (mean ± SD: 9.83 ± 3.9 x 10–2 mm vs 6.3 ± 2.8 x 10–2 mm, P < 0.05). Fourty-eight hours after challenge the ear thickness was still higher in HDC–/– mice compared to wild-type mice, but the difference was not significant between the two groups (mean ± SD: 12.4 ± 3.3 x 10–2 mm vs 7.2 ± 5.2 x 10–2 mm, P > 0.05) (Fig. 1).



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Fig. 1. DNFB-induced increase of ear thickness in HDC–/– and wild-type mice. Contact hypersensitivity response was challenged with 0.2% DNFB in sensitized HDC–/– (n = 6) and wild-type mice (n = 4). Ear swelling was measured 24 (A) and 48 (B) h after challenge with a micrometer. Data are presented as the mean ± SD. *P = 0.023 DNFB-treated HDC–/– vs DNFB-treated wild-type mice.

 
The percentages of CD4+ Th and CD8+ Tc cells were lower, those of CD45R+ B cells were higher in the DLNs of HDC–/– mice
The axillary and inguinal DLNs were excised 48 h after the DNFB challenge and cell suspensions were prepared for phenotypic analysis by flow cytometry. No significant difference was observed between the total number of the DLN cells in the DNFB treated HDC–/– (mean ± SD: 49.54 ± 15.87 x 106 vs 43.25 ± 7.11 x 106, P > 0.05) and wild-type mice. The percentages of CD3+ T (45.4 ± 3.9% vs 61.2 ± 4.1%), CD4+ Th (37.7 ± 3.9% vs 44.6 ± 2.5%) and CD8+ Tc (11.7 ± 1.7% vs 19.2 ± 3.4%) were significantly lower in the HDC–/– mice. In contrast, the percentage of CD45R+ B cells (39.8 ± 5.1% vs 28.5 ± 6.4%) was significantly higher in the HDC–/– mice than in the wild-type mice (Fig. 2). The percentages of granulocytes (6.1 ± 1.7% vs 6.3 ± 1.3%) and macrophages (2.1 ± 1.7% vs 1.4 ± 1.0%) did not differ in the two groups. Similar differences were seen in the cell composition of the axillary and inguinal lymph nodes of untreated HDC–/– and wild-type mice. The percentages of the different cell subpopulations did not significantly differ from those found in the appropriate DNFB treated groups (data not shown).



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Fig. 2. Distribution of lymphocyte subpopulations in DLN. The DLN were excised 48 h following the DNFB challenge and analysed by flow cytometry. **P < 0.001 DNFB-sensitized HDC–/– vs DNFB-sensitized wild-type mice.

 
The number of infiltrating cells was higher in the ear specimens of HDC–/– mice
Histologic sections were made from the ears 24 and 48 h after challenge. In contrast to the acetone/olive treated ear specimens, in the DNFB painted ears of both HDC–/– and wild-type mice a cellular infiltrate and edema were seen. The majority of the infiltrating cells were neutrophil granulocytes and mononuclear cells in both DNFB treated groups at 24 and 48 h after challenge, but the number of infiltrating cells and the degree of edema was higher in the HDC–/– mice (Fig. 3). In ear samples taken 24 or 48 h after DNFB painting, mast cells were stained with toluidine blue. At these time points, no difference was detected in the number of mast cells in the histologic sections of DNFB challenged and acetone/olive treated ears of either HDC–/– or in wild-type mice (data not shown).



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Fig. 3. Hematoxylin–eosin staining of DNFB-treated ears 24 h after challenge. No inflammation was observed in the ears of DNFB-sensitized wild-type (A) and HDC–/– mice (B) following acetone/olive oil treatment. Neutrophil granulocytes and macrophages were the dominant cell types in the dermis 24 h after DNFB challenge both in the HDC–/– (D) and in the wild-type mice (C). The degree of edema and the number of infiltrating cells were higher in the HDC–/– mice (D) compared to the wild-type mice (C).

 
Strong CD45+ leukocyte infiltration was observed in the ears of HDC–/– mice
In order to characterize the phenotype of the infiltrating cells, 3 µm cryostat sections were prepared and the ABC method was used for the immunohistologic staining. We observed a significantly higher percentage of CD45+ leukocytes in the dermis of the ears of the HDC–/– mice than in that of wild-type mice. The number of CD3+ T cells was not increased in the DNFB painted ears compared to the acetone treated ones in either group (Fig. 4).



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Fig. 4. Immunohistological detection of CD45+ leukocytes and CD3+ T cells in ear samples. After DNFB challenge, a strong CD45+ leukocyte infiltration was found in HDC–/– mice (B) compared to wild-type mice (A). The DNFB-painted ears of neither HDC–/– (D), nor wild-type mice (C) showed elevated CD3+ T cell numbers. Normal rat serum was used as negative control for staining the DNFB-treated ears of HDC–/– (F) and wild-type mice (E).

 
IL-2, IFN-{gamma}, TNF-{alpha} and IL-4 mRNA expressions were examined by Q-RT–PCR
Quantitative relationship between the level of gene expression and relative fluorescence data was demonstrated for each examined cytokine gene. Dilution series of a cDNA was used as template and standard curves were generated where the relative fluorescence data was shown as a function of rate of dilution. The correlation coefficient was >0.9 in each of the examined genes (data not shown), suggesting that the reaction conditions applied resulted in quantitative RT–PCR data. Standard curves showed linearity, indicating a quantitative relationship between the relative gene expression and relative fluorescence data (data not shown). The expression of IL-2, IFN-{gamma}, TNF-{alpha} and IL-4 genes was examined by optimized Q-RT–PCR reactions in the ear samples obtained at 24 and 48 h after challenge.

In wild-type mice, IL-2 mRNA was undetectable; in contrast, HDC–/– mice constitutively expressed a detectable level of IL-2 mRNA. In the HDC–/– mice, the DNFB treatment caused a >8-fold increase in the level of IL-2 mRNA 24 h after challenge, however, the quantity of IL-2 mRNA decreased 48 h after challenge. In contrast, in wild-type mice, IL-2 mRNA was not detected 24 h after challenge and it reached a detectable level 48 h after challenge (Fig. 5A).



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Fig. 5. Q-RT–PCR analysis of IL-2, IFN-{gamma} and TNF-{alpha} mRNA expressions in ear samples. HDC–/– mice constitutively express higher levels of IL-2 and TNF-{alpha} cytokine genes than wild-type mice. The mRNA of these cytokines was not detectable in wild-type mice before challenge and 24 h after challenge. The DNFB treatment caused an increase in IL-2 (A), IFN-{gamma} (B) and TNF-{alpha} (C) mRNA expression in HDC–/– mice 24 h after challenge. In contrast, 48 h after challenge, the IL-2, IFN-{gamma} and TNF-{alpha} mRNA level decreased in HDC–/– mice and showed an increase in wild-type mice.

 
The IFN-{gamma} mRNA level showed a significantly higher increase in HDC–/– mice than in wild-type mice 24 h after challenge. Forty-eight hours after challenge, the IFN-{gamma} mRNA level decreased in HDC–/– mice while increasing in wild-type mice (Fig. 5B).

The HDC–/– mice constitutively expressed a detectable level of TNF-{alpha}, while in wild-type mice TNF-{alpha} was undetectable. The increase in TNF-{alpha} expression was 7-fold in HDC–/– mice 24 h after the DNFB treatment, and ~3.5-fold higher 48 h after challenge. In the wild-type mice, TNF-{alpha} mRNA was not detected 24 h after the treatment, and showed an increase 48 h after challenge (Fig. 5C).

The expression of IL-4 mRNA reached a detectable level in HDC–/– mice but not in wild-type mice. The DNFB treatment of HDC–/– mice produced a moderate increase of IL-4 mRNA expression 24 h after challenge, and the increase in IL-4 mRNA expression was 5-fold 48 h after the treatment compared to the 24 h data. In the wild-type mice, no IL-4 mRNA was demonstrated 24 h after challenge, but a detectable amount of mRNA appeared 48 h after the DNFB treatment (Fig. 6).



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Fig. 6. Q-RT–PCR analysis of IL-4 mRNA expression in ear samples. The DNFB treatment of HDC–/– mice produced a moderate increase of IL-4 mRNA expression 24 h after challenge and a strong increase 48 h after challenge. In the wild-type mice, IL-4 mRNA was not detected 24 h after challenge, but it was demonstrated 48 h after challenge.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Histamine is an early messenger in inflammatory reactions. It regulates the immune response by enhancing Th2 (IL-4, IL-10) and by inhibiting Th1 (IL-2, IFN-{gamma}, TNF-{alpha}) cytokine production (4,5,34). In the present study, we investigated in HDC–/– histamine-deficient mice whether histamine has a regulatory role in DNFB-induced CHS and whether the lack of histamine modifies the cytokine profile.

We found that in histamine-deficient mice, DNFB-induced CHS is more intense than in wild-type mice. The DNFB-induced increase of the ear thickness was significantly higher in the HDC–/– mice 24 h after challenge than in wild-type mice. Forty-eight hours after challenge the ear thickness was still higher in HDC–/– mice but the difference was not significant between the two groups. After DNFB challenge, the percentages of CD3+, CD4+ and CD8+ T cells in the DLN of sensitization area were significantly lower, those of CD45R+ B cells were significantly higher in HDC–/– mice than in wild-type mice. Similar differences were found in the DLNs of the untreated HDC–/– and wild-type mice. Consequently, these differences do not seem to be due to the DNFB treatment, but they are rather associated with the lack of histamine in HDC–/– mice.

The inflammatory reaction in the ear skin of the mice was also studied. We found that 24 h after challenge the number of infiltrating cells and the degree of edema was higher in the HDC–/– mice than in the wild-type mice. In hapten challenge sites, neutrophils recruit CD8+ T cells that subsequently produce cytokines mediating the hypersensitivity response (21,35). Using HDC–/– mice, Hirasawa et al. (6) found that histamine plays a negative regulatory role for the neutrophil infiltration via H2R receptor in allergic inflammation. It has been reported that in the skin of HDC–/– mice, the expression of H1R and H2R receptors is very sensitive to histamine levels and both receptors are downregulated in the skin of HDC–/– mice (36). These results suggest that histamine might inhibit neutrophil infiltration in wild-type mice via H2R receptors and the lack of histamine favors a strong granulocyte and macrophage infiltration in HDC–/– mice.

We observed an increase of the ear thickness and relatively few infiltrating cells in wild-type mice 24 h after challenge. In the early phase of elicitation of CHS (3–24 h after challenge), release of serotonin and TNF-{alpha} from mast cells and platelets results in an increased vascular permeability and tissue swelling (1416). These data indicate that the increase of the ear thickness is mainly due to edema formation 24 h after challenge.

We also observed that HDC–/– mice constitutively express higher levels of IL-2, TNF-{alpha} and IL-4 mRNAs than wild-type mice. These findings suggest that endogenous histamine downregulates the production of IL-2, TNF-{alpha} and IL-4. It is known that CD8+ Tc1 cells mainly produce Th1 type cytokines. In our study, the DNFB treatment caused higher levels of Th1 cytokine mRNAs (IL-2, IFN-{gamma}, TNF-{alpha}) in HDC–/– mice 24 and 48 h after challenge, and a higher level of Th2 cell cytokine (IL-4) mRNA 48 h after challenge than in wild-type mice.

Challenge with antigen in sensitized mice induces local recruitment of T cells. These antigen-specific T cells produce inflammatory cytokines, which induce ear swelling and other inflammatory processes in the later phase of elicitation (48–72 h after challenge). We observed a very early Th1 cytokine response in HDC–/– mice, followed by the increased levels of IL-2, IFN-{gamma} and TNF-{alpha} mRNAs 24 h after DNFB challenge. In these mice the high levels of Th1 cytokines might contribute to the very early increase of the ear thickness and the inflammatory response demonstrated by immunohistology.

We demonstrated that in the early phase of elicitation, the ear thickness was greater in HDC–/– mice than in wild-type mice. Twenty-four hours after challenge, the levels of Th1 cytokine mRNAs were significantly higher in the ear samples of histamine-deficient mice compared to wild-type mice. These data suggest that histamine might have a suppressive effect on the production of Th1 cytokines and, consequently, on the limitation of the inflammatory response. In the later phase of elicitation there was no significant difference in the ear swelling in the two groups. Forty-eight hours after challenge, in wild-type mice a significant increase of Th1 cytokine mRNAs was observed, which was comparable with that seen in HDC–/– mice at 24 h after challenge. The levels of Th1 cytokine mRNAs in HDC–/– mice 48 h after challenge were higher than those observed in wild-type mice, however, in the HDC–/– mice, significantly increased IL-4 levels were also demonstrated. Recent studies have shown that both Th1 and Th2 T cells are involved in the regulation of contact hypersensitivity. IL-4 is a Th2 cytokine that plays an important role during the elicitation phase of CHS, and has a role in the mediation of inflammation (25).

Ohtsu et al. (31) has recently reported that in the CHS response, the ear thickness of HDC–/– mice was not significantly different from that of wild-type mice. However, in their experiments another sensitizing agent, trinitrochlorobenzene was used in very high concentrations. We showed, using flow cytometry, immunohistology, and Q-RT–PCR, that DNFB induced a more intense inflammation in HDC–/– mice than in wild-type mice. The discrepancy between their and our results might be explained by the different experimental conditions.

Our data suggest that histamine has an important role both in the early and in the later phase of CHS reaction. The lack of histamine seems to be responsible for a very intense Th1 type response in the early phase and also for a strong Th2 response in the late phase of CHS.

Histamine is known to inhibit Th1 lymphocyte functions such as production of IL-2, IFN-{gamma} via H2R receptors, and to enhance Th1-type responses by triggering the H1R receptors (8,37). Fitzsimons et al. (36) demonstrated that in the skin of HDC–/– mice the H1R and H2R receptors are downregulated, which might be due to the prolonged histamine deficiency. We found a very early and high Th1 cytokine response after antigen challenge that might be caused by histamine deficiency. These data indicate that endogenous histamine can downregulate the CHS reaction via H2R receptor in wild-type mice. The lack of histamine causes a downregulation of H2R receptors in HDC–/– mice, thereby leading to a higher Th1 cytokine response compared to wild-type mice. These results suggest that in the histamine-deficient mice the Th1/Th2 balance is modulated towards Th1 dominancy.

In our study, we demonstrated that histamine is involved in the regulation of delayed type hypersensitivity. Using histamine-deficient mice we showed that histamine plays a suppressive immunoregulatory role in the DNFB-induced CHS response.


    Acknowledgements
 
This work was supported by the grants NKFP 1A/0012/2002, OTKA TS 0044826, OTKA T042738, Hungarian Ministry of Health (ETT 408 05/2000) and Netherlands Organization for Scientific Research (NWO 047–003040). Márta Széll was supported by the Bolyai Foundation of the Hungarian Academy of Sciences.


    Abbreviations
 
AC   acetone
CHS   contact hypersensitivity
DNFB   dinitrofluorobenzene
DLN   skin-draining lymph nodes
HDC   histidine decarboxylase
H1R   histamine receptor type 1
H2R   histamine receptor type 2
Q-RT–PCR   quantitative reverse transcription polymerase chain reaction

    Notes
 
Transmitting editor: I. Pecht

Received 24 November 2003, accepted 28 September 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Kubo, Y. and Nakano, K. 1999. Regulation of histamine synthesis in mouse CD4+ and CD8+ T lymphocytes. Inflamm. Res. 48:149.[CrossRef][ISI][Medline]
  2. Laszlo, V., Rothe, G., Hegyesi, H., Szeberenyi, J. B., Orso, E., Schmitz, G. and Falus, A. 2001. Increased histidine decarboxylase expression during in vitro monocyte maturation; a possible role of endogenously synthesised histamine in monocyte/macrophage differentiation. Inflamm. Res. 50:428.[ISI][Medline]
  3. Szeberenyi, J. B., Pallinger, E., Zsinko, M. et al. 2001. Inhibition of effects of endogenously synthesized histamine disturbs in vitro human dendritic cell differentiation. Immunol. Lett. 76:175.[CrossRef][ISI][Medline]
  4. Igaz, P., Novak, I., Lazar, E., Horvath, B., Heninger, E. and Falus, A. 2001. Bidirectional communication between histamine and cytokines. Immunol. Res. 50:123.
  5. Falus, A. and Meretey, K. 1992. Histamine:an early messenger in inflammatory and immune reactions. Immunol. Today 13:154.[CrossRef][ISI][Medline]
  6. Hirasawa, N., Ohtsu, H., Watanabe, T. and Ohuchi, K. 2002. Enhancement of neutrophil infiltration in histidine decarboxylase-deficient mice. Immunology 107:217.[CrossRef][ISI][Medline]
  7. Novak, I. and Falus, A. 1997. Molecular biology and role of histamine in physiological and pathological reactions. A review. Acta Biol. Hung. 48:385.[ISI][Medline]
  8. Jutel, M., Watanabe, T., Klunker, S. et al. 2001. Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors. Nature 413:420.[CrossRef][ISI][Medline]
  9. Askenase, P. W. 1998. Effector and regulatory molecules and mechanisms in delayed type hypersensitivity. In Middleton, E., Ellis, E. F., Yunginger, J. W., Reed, Ch. E., Adkinson, N. F. and Busse, W. W., eds, Allergy. Principles & Practice. Fifth edition.
  10. Gerberick, G. F., Cruse, L. W., Miller, C. M. and Ridder, G. M. 1999. Selective modulation of B-cell activation markers CD86 and I-Ak on murine draining lymph node cells following allergen or irritant treatment. Toxicol. Appl. Pharmacol. 159:142.[CrossRef][ISI][Medline]
  11. Fujisawa, H., Kondo, S., Wang, B., Shivji, G. M. and Sauder, D. N. 1996. The role of CD4 molecules in the induction phase of contact hypersensitivity cytokine profiles in the skin and lymph nodes. Immunology 89:250.[CrossRef][ISI][Medline]
  12. Gorbachev, A. V. and Fairchild, R. L. 2001. Induction and regulation of T-cell priming for contact hypersensitivity. Crit. Rev. Immunol. 21:451.[ISI][Medline]
  13. Wang, B., Feliciani, C., Howell, B. G., Freed, I., Cai, Q., Watanabe, H. and Sauder, D. N. 2002. Contribution of Langerhans cell-derived IL-18 to contact hypersensitivity. J. Immunol. 168:3303.[Abstract/Free Full Text]
  14. Askenase, P. W., Bursztajn, S., Gershon, M. D. and Gershon, R. K. 1980. T cell-dependent mast cell degranulation and release of serotonin in murine delayed-type hypersensitivity. J. Exp. Med. 152:1358.[Abstract]
  15. Van Loveren, H., Meade, R. and Askenase, P. W. 1983. An early component of delayed-type hypersensitivity mediated by T cells and mast cells. J. Exp. Med. 157:1604.[Abstract]
  16. Askenase, P. W., Geba, G. P., Levin, J., Ratzlaff, R. E., Anderson, G. M., Ushio, H., Ptak, W. and Matsuda, H. 1995. A role for platelet release of serotonin in the initiation of contact sensitivity. Int. Arch. Allergy Immunol. 107:145.[ISI][Medline]
  17. Geba, G. P., Ptak, W., Anderson, G. M., Paliwal, V., Ratzlaff, R. E., Levin, J. and Askenase, P. W. 1996. Delayed-type hypersensitivity in mast cell-deficient mice: dependence on platelets for expression of contact sensitivity. J. Immunol. 157:557.[Abstract]
  18. Askenase, P. W. and Tsuji, R. F. 2000. B-1 B cell IgM antibody initiates T cell elicitation of contact sensitivity. Curr. Top. Microbiol. Immunol. 252:171.[ISI][Medline]
  19. Enk, A. H. 1997. Allergic contact dermatitis: understanding the immune response and potential for targeted therapy using cytokines. Mol. Med. Today 3:423.[CrossRef][ISI][Medline]
  20. Akiba, H., Kehren, J., Ducluzeau, M. T., Krasteva, M., Horand, F., Kaiserlian, D., Kaneko, F. and Nicolas, J. F. 2002. Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis. J. Immunol. 168:3079.[Abstract/Free Full Text]
  21. Martín, A., Gallino, N., Gagliardi, J., Ortiz, S., Lascano, A. R., Diller, A., Daraio, M. C., Kahn, A., Mariani, A. L. and Serra, H. M. 2002. Early inflammatory markers in elicitation of allergic contact dermatitis. BMC Dermatology 2:9.[CrossRef][Medline]
  22. Kondo, S. and Sauder, D. N. 1995. Epidermal cytokines in allergic contact dermatitis. J. Am. Acad. Dermatol. 33:786.[CrossRef][ISI][Medline]
  23. Xu, H., DiIulio, N. A. and Fairchild, R. L. 1996. T cell populations primed by hapten sensitization in contact sensitivity are distinguished by polarized patterns of cytokine production: interferon gamma-producing (Tc1) effector CD8+ T cells and interleukin (Il) 4/Il-10-producing (Th2) negative regulatory CD4+ T cells. J. Exp. Med. 183:1001.[Abstract]
  24. Ulrich, P., Grenet, O., Bluemel, J., Vohr, H. W., Wiemann, C., Grundler, O. and Suter, W. 2001. Cytokine expression profiles during murine contact allergy: T helper 2 cytokines are expressed irrespective of the type of contact allergen. Arch. Toxicol. 75:470.[CrossRef][ISI][Medline]
  25. Watanabe, H., Unger, M., Tuvel, B., Wang, B. and Sauder, D. N. 2002. Contact hypersensitivity: The mechanism of immune responses and T cell balance. J. Interferon. Cytokine Res. 22:407.[CrossRef][ISI][Medline]
  26. Belsito, D. V., Kerdel, F. A., Potozkin, J. and Soter, N. A. 1990. Cimetidine-induced augmentation of allergic contact hypersensitivity reactions in mice. J. Invest. Dermatol. 94:441.[Abstract]
  27. Griswold, D. E., Alessi, S., Badger, A. M., Poste, G. and Hanna, N. 1986. Differential sensitivity of T suppressor cell expression to inhibition by histamine type 2 receptor antagonists. J. Immunol. 137:1811.[Abstract/Free Full Text]
  28. Grob, J. J., Castelain, M., Richard, M. A., Bonniol, J. P., Beraud, V., Adhoute, H., Guillou, N. and Bonerandi, J. J. 1998. Antiinflammatory properties of cetirizine in a human contact dermatitis model. Clinical evaluation of patch tests is not hampered by antihistamines. Acta Derm. Venereol. 78:194.[CrossRef][ISI][Medline]
  29. Ohtsu, H., Tanaka, S., Terui, T. et al. 2001. Mice lacking histidine decarboxylase exhibit abnormal mast cells. FEBS Lett. 502:53.[CrossRef][ISI][Medline]
  30. Wiener, Z., Andrasfalvy, M., Pallinger, E., Kovacs, P., Szalai, C., Erdei, A., Toth, S., Nagy, A. and Falus, A. 2002. Bone marrow-derived mast cell differentiation is strongly reduced in histidine decarboxylase knockout, histamine-free mice. Int. Immunol. 14:381.[Abstract/Free Full Text]
  31. Ohtsu, H., Kuramasu, A., Tanaka, S. et al. 2002. Plasma extravasation induced by dietary supplemented histamine in histamine-free mice. Eur. J. Immunol. 32:1698.[CrossRef][ISI][Medline]
  32. Par, G., Szekeres-Bartho, J., Buzas, E., Pap, E. and Falus, A. 2003. Impaired reproduction of histamine deficient (histidine-decarboxylase knockout) mice is caused predominantly by a decreased male mating behavior. Am. J. Reprod. Immunol. 50:152[CrossRef][ISI][Medline]
  33. Jánossy, T., Baranyi, L., Knulst, C. A., Vizler, C., Benner, R. and Vegh, P. 1993. MHC-specific graft-protective and delayed-type hypersensitivity (DTH) suppressive activity of a CD4+CD8+, {alpha}ß T cell receptor (TCR) positive lymphoma isolated from a tolerant mouse. Immunobiology 188:172.[ISI][Medline]
  34. Horvath, B. V., Szalai, C., Mandi, Y., Laszlo, V., Radvany, Z., Darvas, Z. and Falus, A. 1999. Histamine and histamine-receptor antagonists modify gene expression and biosynthesis of interferon gamma in peripheral human blood mononuclear cells and in CD19-depleted cell subsets. Immunol. Lett. 70:95.[CrossRef][ISI][Medline]
  35. Zhang, L. and Tinkle, S. S. 2000. Chemical activation of innate and specific immunity in contact dermatitis. J. Invest. Dermatol. 115:168.[Abstract/Free Full Text]
  36. Fitzsimons, C. P., Lazar-Molnar, E., Tomoskozi, Z., Buzas, E., Rivera, E. S. and Falus, A. 2001. Histamine deficiency induces tissue-specific down-regulation of histamine H2 receptor expression in histidine decarboxylase knockout mice. FEBS Lett. 508:245.[CrossRef][ISI][Medline]
  37. Ohuchi, Y., Ohtsu, H., Sakurai, E., Yanai, K., Ichikawa, A., Radvany, Z., Darvas, Z., Falus, A. and Watanabe, T. 1998. Induction of histidine decarboxylase in type 2 T helper lymphocytes treated with anti-CD3 antibody. Inflamm. Res. 47(Suppl. 1):S48.[CrossRef][ISI][Medline]