Delayed-type hypersensitivity reactions to nominal protein antigens and to environmental allergens: similarities and differences

ARTICLE

When an adaptive immune response occurs in an exaggerated or inappropriate form causing tissue damage, the term hypersensitivity is applied. Hypersensitivity is manifested on second contact with the particular antigen evoking hypersensitivity. Coombs and Gells [1] originally proposed a "classification of allergic reactions responsible for clinical hypersensitivity and diseases" and described four types of hypersensitivity reactions. However in 1968, the under-lying immune mechanisms were not clearly identified, in particular in terms of lymphocyte subpopulations, activation markers and cytokines. Nevertheless, this first classification, although elementary, was applicable to different clinical situations. Indeed immunological mechanisms leading to tissue inflammation may involve any of these classical effector mechanisms. Cutaneous hypersensitivity reactions have been well studied in animal models, but fewer studies are available in humans. Four types of late-onset dermal reactions to injected antigen have been described in humans [2]. These reactions include delayed type hypersensitivity (DTH), cutaneous basophil hypersensitivity, Arthus reactions, and late phase allergic reactions.

This review will focus on the classical type of DTH represented by the tuberculin reaction, and on a less classical type represented by the allergen-induced late phase reaction (LPR) which occurs in atopic patients. DTH and LPR are both mediated by antigen-specific T cells directed to classical nominal proteins (DTH) or to environmental protein allergens (LPR). Although cutaneous LPR and DTH have unique characteristics they also have features in common.

Pathophysiological mechanisms

Tuberculin-induced delayed type hypersensitivity

This form of T cell-mediated hypersensitivity was initially described by Koch, who observed that patients with tuberculosis reacted with fever and generalized sickness following a subcutaneous injection of tuberculin. This reaction was accompanied by an area of induration and swelling at the site of injection. DTH reactions may follow tuberculin challenge and begin as local itching about 12 hrs after intradermal challenge. The injection site subsequently develops into an area of local, sometimes painful, induration that peaks in intensity from 24 to 72 hrs prior to resolution.

Role of antigen-presenting cells, T cells and other cell types.

Macrophages start to accumulate around dermal vessels at 12 hrs, their numbers increasing up to 72 hrs. Cells of the macrophage lineage are probably the main antigen-presenting cells in the tuberculin hypersensitivity reaction, but the finding of CD1⁺ cells in the dermal infiltrate suggests that Langerhans cells or indeterminate dendritic cells may be involved. Indeed CD1⁺ Langerhans cells are found in the dermal infiltrate at 48 hrs [3]. However, in mice, depletion of epidermal Langerhans cells does not alter the DTH response [4]. After encountering the antigen, the few antigen-specific T cells will become activated and secrete cytokines providing the amplification signals which result in localized inflammatory responses.

Twelve hours after intradermal tuberculin challenge, T lymphocytes are present at perivascular sites and this infiltrate, which extends outwards and disrupts the collagen bundles of the dermis, increases to a peak at 48 hrs. CD4⁺ cells outnumber CD8⁺ cells by about 2:1. T cells are activated as shown by an increase in CD25 and HLA-DR expression [5]. Infiltrating macrophages may also express HLA-DR as well as overlying keratinocytes which express it 48-96 hrs after the appearance of the lymphocytic infiltrate [6]. T cells mediating tuberculin-induced DTH are MHC class II restricted in humans, at least as assessed in vitro [7-8]. In mice DTH reactions are elicited by CD4⁺ cells with apparent downregulatory effects on CD8⁺ cells [9]. Because patients with late stage AIDS can still develop contact dermatitis (dependent on CD8) but not tubeculin-type DTH [10], this concept may also apply to humans.

Neutrophils and eosinophils are rarely found in human DTH [5]. While no specific role has been assigned to mast cells in the human tuberculin reaction, it is of interest to note that in this situation they express HLA-DR [11] which is inducible by IFN-gamma [12], and that they can, in vitro, present tuberculin to PPD-specific T cell lines [13].

Traditionally, it is believed that antigen-specific, primed T cells which carry skin homing receptors (such as the cutaneous lymphocyte antigen), constantly patrol through the skin, and encounter their relevant antigen within the skin. Although it has been shown that a single antigen-specific T cell is enough to induce a DTH response [14], it is unlikely that sufficient numbers of antigen-specific T cells are present at any given time point in any part of the skin to elicit DTH responses. Alternatively, antigen-specific T cells could be recruited through the direct proinflammatory effect of tuberculin on resident cells such as macrophages [15] leading to cytokine and chemokine production.

Role of cytokines and chemokines.

The dependence of the DTH reaction upon IFN-g is well known in rodents, but it is only recently that its participation was shown in human cutaneous tuberculin-induced DTH. Using in situ hybridization a predominent Th-1 type cytokine profile was evidenced with a strong expression of IFN-g and IL-2 while few IL-4 and IL-5 mRNA positive cells were observed [16] (see Fig. 1 for representative in situ hybridization experiments). Other cytokines like TGF b also have a role in the late initiation of a fibrotic response after tuberculin injection [17]. In contrast, IL-10, a prototypic inhibitor of Th-1 type cytokines, is found at low levels in tuberculin reactions [18] in particular compared to cutaneous LPR [19]. IL-10 has been shown to inhibit DTH reactions in mice through inhibition of IL-2 and IFN-gamma production [20]. A few studies have looked at the involvement of chemokines in human DTH reactions. One showed the implication of IP-10 [21], known to act through CXCR3 which is expressed on Th1 type cells [22]. Another demonstrated the presence of RANTES in DTH, in particular on macrophages and endothelial cells [23] which can also attract recently activated T cells xpressing CCR5, expressed on Th-1 cells. In animal models of DTH, several chemokines have been involved, such as IL-8 [24] and MCP-1 [25]. Their respective inhibition antagonizes the DTH reaction. The importance of MCP-1 has been recently indirectly confirmed in CCR2 knock-out mice. These mice, which lack the MCP-1 receptor, have a defect in both tuberculin-induced DTH responses and in Th-1 type cytokine production [26].

Altogether, the release of chemokines and proinflammatory cytokines may form an initial non specific inflammatory stimulus, which then would allow antigen-specifc T cells to accumulate at the site of inflammation, to become activated, and to amplify the whole inflammatory reaction. The sharing of chemokine receptor would promote the encounter of antigen-specific T cells with different effector cells. Thus the common expression of CCR1 and CCR5 on Th-1 cells and monocytes which represent the precursors of macrophages and dendritic cells, would allow their joint recruitment at the site of DTH reactions.

Role of adhesion molecules.

The recruitment from blood of cells able to penetrate the site of the DTH reaction necessitates the expression of adhesion molecules on the vascular endothelium. Some studies in humans have looked at the expression of such molecules after the cutaneous injection of tuberculin. Following PPD injection, the expression of endothelial E-selectin, VCAM-1 and keratinocyte ICAM-1 is observed [27]. Inhibition experiments in in vivo animal models have shown that leukocyte recruitment in cutaneous DTH is dependent upon E- and P- selectins [28, 29], which is consistent with the recent description of these molecules as mediating the recruitment of Th-1 but not Th-2 cells into the skin [30]. In this context it is of interest that some chemokine receptors may play a role in adhesion, such as CCR1 able to bind P- and E- selectin as alternative ligands.

Allergen-induced late phase reactions

The recognition and description of LPRs actually dates back over a century and was reported by Blackley in 1873 [31] describing late symptoms evolving several hours after exposure to grass pollen. Following allergen challenge in the skin, an IgE-dependent wheal and flare reaction develops almost immediately and is characterized by a central area of pale swelling surrounded by a halo of erythema. This macroscopic response which is intensely pruritic, peaks in 10 to 15 min and will, with a suffisant amount of allergen, evolve into a LPR, characterized by burning, pruritus, erythema, induration and warmth. Cutaneous LPRs generally peak at 6 to 8 hrs and may resolve by 48 hrs.

Role of mast cells, T cells and other cell types.

A number of studies have demonstrated that cutaneous LPRs can be mediated by IgE antibody, in particular Dolovich et al. who reproduced it by injecting antihuman IgE F(ab')2 fragments [32]. The acute reaction, through Fc epsilonRI crosslinking, is associated with mast cell degranulation and mediator release such as histamine but also preformed cytokines which can initiate the events in LPR. Skin blister fluid studies have shown that the intensity of the LPR is correlated with the initial quantity of released histamine [33] and PGD2 [34] and that a sustained production of histamine is observed during the development of the LPR, probably related to basophils [35]. Histamine is only a marker of other inflammatory responses because histamine itself does not induce LPR. Although the immediate release of cytokines after IgE-dependent stimulation, such as TNF-alpha, IL-3, IL-4 and IL-5 by mast cells, would be a good candidate as an LPR trigger, the difficulty of detecting these mediators in skin blisters has limited this type of study [36]. More recently mast cells have also been shown to produce a number of chemokines such as MIP-1alpha, eotaxin and RANTES that could contribute to the attraction of the effector cells of the allergic reaction [37-39]. Other cell types in cutaneous LPR have been shown to bear high affinity receptors for IgE such as Langerhans cells, macrophages, and eosinophils and may be involved in the development of cutaneous LPRs through mediator release but also through antigen presentation function [40].

Skin biopsies performed at different time points after allergen challenge in atopic subjects developing an LPR have shown evidence of infiltration and activation of T cells and eosinophils. A substantial number of CD3 positive cells, as evidenced by immunocytochemistry, are observed close to the dermal capillaries, mainly of the CD4⁺ subpopulation. Activation markers are expressed such as the IL-2 receptor CD25, as well as activated eosinophils [41]. CD4⁺ cells are thought to be major actors in allergic reactions, in particular through the release of cytokines involved in the activation of effector cells. Their importance has been recently confirmed by the efficacy of humanized anti-CD4 monoclonal antibodies in asthma [42]. Antigen presentation to CD4⁺ lymphocytes has been shown to be MHC class II restricted for a number of allergens such as dermatophagoides pteronyssinus [43, 44]. The role of CD8⁺ cells remains hypothetical, although their ability to produce IFN-gamma would suggest a negative regulatory role in the allergic inflammatory reaction through inhibition of IgE synthesis and Th-2 function. Eosinophils are another hallmark of allergic reactions, and are thought to be responsible for the local damage associated with these reactions.

Role of cytokines and chemokines.

The role of cytokines in the development of the LPR was recognized in the nineties when in situ hybridization studies showed the predominant expression of IL-4, known for its induction of IgE synthesis, of IL-5, a differentiation and activation factor of eosinophils, of IL-3 and GM-CSF, also active on eosinophils. However, no expression of IL-2 and IFN-gamma was observed, therefore suggesting a preferential Th-2 type activation in cutaneous LPR [45]. (Fig. 1). Chemokines are strong granulocyte and mononuclear cell chemoattractants, and it was next shown that RANTES, and MCP-3, active on eosinophils and memory T cells, were strongly expressed at the site of cutaneous LPR [46]. Expression of MCP-3 paralleled the eosinophil infiltration while RANTES expression followed the kinetics of T cell and macrophage infiltration, suggesting their implication in the recruitment of the different cell populations. Other chemokines such as IL-8 and MCP-1, more active respectively on neutrophils and monocytes, have been found in blister models of LPR [47]. Therefore, the initial release of preformed mediators by allergen-triggered mast cells could lead to the activation of resident cells and release of chemokines and cytokines preferentially activating and recruiting Th-2 cells and eosinophils. In this respect the recent description of locally produced eotaxin in late phase asthmatic reactions[48, 49] is of potential interest in the context of the expression of its receptor CCR3 on both eosinophils and Th-2 cells [50].

Role of adhesion molecules.

The development of the inflammatory process involves a variety of changes including increased vascular permeability, expression of endothelial cell adhesion molecules like E-selectin, ICAM and VCAM, interaction between these molecules and their counter ligands on leukocytes allowing their margination, firm adhesion and transendothelial migration. Up-regulation of E-selectin has been clearly demonstrated in sequential biopsies obtained from allergen-induced LPR reactions and shown to be TNF-alpha dependent by using skin cultures [51]. This model showed that resident cells in the skin rather than infiltrating leukocytes appeared to be the source of cytokines that mediated endothelial activation. All these events are closely intricated in the release of mediators. Thus IL-4, released after IgE-dependent stimulation of mast cells, will upregulate VCAM-1 on endothelial cells. Eosinophils through their counter ligand VLA-4 will interact with VCAM-1 allowing their transmigration to the tissue compartment. In this situation too, chemokines may have a role to play, indeed it has been shown that thay are required for the interaction of VLA-4 with fibronectin, which is important for cell migration into tissue.

Similarities and differences

At a first glance, DTH and LPR would appear quite different. They display completely different clinical aspects, kinetics and cellular infitration and they have opposite polarized cytokine patterns (see Table I for the summary of their differences). One of the main differences is linked to the type of immunogen used, PPD and allergen, known respectively to favour a Th-1 and a Th-2 response. Resident cells at the site of antigen exposure might also contribute to differences between these responses. By virtue of their specific cytokine and chemokine secretion pattern, allergen-activated mast cells may determine the microenvironment favouring a Th-2 response while in DTH macrophages will be the cell type favouring a Th-1 response. The pathophysiological consequences are also contrasted, DTH reactions being usually linked to protection, while LPR appears as a deleterious reaction.

In order to compare these two types of reaction more closely, and to eliminate as much as possible the influence of genetic factors and inter-individual variations, both reactions were elicited simultaneously in the skin of the same atopic subjects, with a comprehensive time course study starting from 1 to 96 hrs post-antigen challenge [52]. Biopsies were taken at different time points and processed for immunohistochemistry and in situ hybridization.

Comparison of the cellular infiltration and cytokine profile in DTH and LPR

Clinically, as classically described, the mean size of the LPR increased up to 6 hrs after allergen challenge and then decreased progressively. In contrast, in DTH, no change in mean reaction size was evident until 24 hrs after tuberculin injection, then it increased reaching a plateau between 48 and 96 hrs.

The kinetics study showed that in the LPR the cellular infiltration of the allergen-injected site started very early from 1 to 3 hrs after challenge for CD4⁺ T cells, eosinophils and neutrophils, peaked at 6 hrs with a predominent influx of these cells, and then decreased. The kinetics of infiltration was different for other cells including macrophages, CD8⁺ T cells and CD25⁺ cells which peaked at 48 hrs. In the DTH reaction elicited at the same moment, the kinetics were completely different with no cellular infiltration before 24 hrs. T cells, neutrophils, macrophages and CD25⁺ cells peaked 48 hrs after the tuberculin injection. Almost no eosinophils were found at any time point (Fig. 2).

In previous studies looking at the cytokine profile of DTH and LPR only the 24 hrs time point was evaluated [16, 45], demonstrating a polarized Th-1 or Th-2 type respectively. Kinetics expression of cytokine mRNA in LPR showed a typical Th-2 profile at the early time points fom 1 to 24 hrs. However surprisingly, an increase in IL-2 and IFN-gamma mRNA expression was observed at 48 hrs, suggesting a late additional Th-1 type component in LPR. Kinetics of DTH showed a typical Th-1 type response at 24 hrs, but also a small increase in IL-4 and IL-5 mRNA expression at 48 hrs, suggesting an additional Th-2 component (Fig. 3).

Thus this study provides some evidence that DTH and LPR are not completely different reactions and that they exhibit components of each other. The late Th-1 component observed in allergen-induced LPR might result from the activation of the CD8⁺ cells recruited at 48 hrs and able to secrete IFN-gamma. Alternatively, the uptake of the allergen by macrophages also preferentially recruited at 48 hrs, and its presentation to T cells, may also favour a Th-1 profile. In contrast, the late Th-2 component observed in the DTH response remains unclear. One could suggest that the late activation of mast cells observed in DTH might favour such a profile although this hypothesis remains completely speculative.

Anyway, the additional Th-1 or Th-2 components seen in each reaction could act as down regulatory effectors of the initial reaction which could explain the close interaction observed between them in some in vivo clinical situations.

Pathological consequences and clinical relevance of DTH and LPR

Cell-mediated immunity is a primary host resistance mechanism against many infectious agents and is responsible for leukocyte recruitment to the infection site. DTH reactions are in vivo correlates of cell-mediated immunity and have long been used to assess the level of immune responsiveness to specific antigens. LPRs appear to contribute substantially to the clinical expression of various allergic diseases including asthma, allergic rhinitis and atopic dermatitis. LPR has become an increasingly powerful tool to investigate allergic mechanisms.

The close interaction observed between these two witnesses of different types of immune responses has been recently highlighted in different studies. In recent decades there has been an increase in the severity and prevalence of atopic disorders in developed countries. One associated factor is the decline of many infectious diseases as the result of improved living standards and immunization programs. A study was carried out comparing the tuberculin-induced DTH reaction and the atopic status of Japanese children. There was a strong inverse correlation between DTH reactions and atopy. Positive tuberculin reactions predicted lower incidence of asthma, lower serum IgE levels, and cytokine profiles biased toward Th-1 type [53] suggesting transregulatory effects between the two immune responses. This hypothesis is further supported by another study where the infection of mice with Mycobacterium bovi-Bacillus Calmette-Guerin suppressed allergen-induced eosinophilia, one of the features of LPRs [54]. Another example is illustrated by specific immunotherapy, an efficient treatment of some allergic diseases, which is effective in decreasing the LPR to allergen. In a study looking at the effects of specific immunotherapy on allergen-induced cutaneous LPR, it was shown that the preferential expression of a Th2 pattern of cytokines in response to allergen exposure was not affected by immunotherapy, but that there was an additional Th1 expression 24 hrs after allergen challenge which could account for T cell tolerance [55]. In other types of specific immunotherapy with more potent allergens a complete shift from a Th2- to Th1-type profile has been observed [56, 57]. This might reflect the anergy observed in antigen-specific T cells during this treatment, leading to a change in cytokine profile as observed at the clonal level [58].

Therefore tuberculin-induced DTH and allergen-induced LPRs are two witnesses of tightly complementary interacting immune responses involved in the regulation of pathological disorders and represent interesting models to study the pathophysiology of, in particular, allergic diseases.

Development of a "pre-human" animal model

However, for obvious ethical reasons, the modulation of such inflammatory reactions are difficult to evaluate in humans. We therefore thought about using an in vivo model of humanized SCID mice. Indeed, these mice, due to their defect in B and T lymphocyte antigen receptors, can accept human grafts, and provide the opportunity to construct human SCID mouse chimeras. Such chimeras were obtained by grafting SCID mice with human skin and by reconstituting them with peripheral blood mononuclear cells (PBMC) from the skin donor [59].

Tuberculin-induced DTH in hu-PBMC-SCID mice grafted with autologous skin

Human donors exhibited positive cutaneous reactions to tuberculin. Skin was obtained during plastic surgery and grafted onto SCID mice. Two full-thickness skin grafts were placed on each mouse. Four weeks after human skin transplantation, the mice were reconstituted intraperitoneally with 15 to 20 million PBMC from the donor. Three weeks after the reconstitution, one graft was injected with tuberculin and the other with the corresponding diluent. Grafts were recovered 72 hrs later (corresponding to the peak of the reaction in humans) and processed for immunohistochemical and in situ hybridization studies.

Tuberculin injection induces a preferential T cell infiltration and an increase in activation markers.

At the site of tuberculin injection as compared with the diluent, a significant infiltration of pan-leukocyte CD45⁺ cells was observed. This increase was mostly accounted for by CD4⁺, CD8⁺ and CD45RO⁺ cells. There was neither additional recruitment of CD68⁺ monocytes-macrophages nor eosinophil infiltration at the site of the tuberculin reaction. The absence of additional macrophage recruitment, contrary to observations seen in the human reaction, might be explained by the delay between the reconstitution and the tuberculin injection (i.e. 3 weeks). Indeed it is known that in reconstitued SCID mice, monocytes have disappeared by that time. Therefore these results suggest that local macrophages already present within the skin are enough to elicit the DTH reaction. A significant increase in two activation markers was observed at the tuberculin injected sites, first in HLA-DR and second in the IL-2 receptor CD25 marker, similarly to the human reaction.

Tuberculin induces a preferential Th-1 type cytokine profile.

The cytokine profile of the reaction was evaluated and a significant increase in IL-2 and IFN-gamma mRNA expressing cells was observed in the tuberculin sites, while no statistical difference was observed for IL-4 and IL-5 as compared with the diluent sites. However at this late time point (72 hrs), the additional Th-2 component was again observed.

These results are similar to those reported in humans, validating the use of this model as a "pre-human" model. Studies on regulatory mechanisms will now be possible, opening new ways to investigate the pathophysiology of such reactions. We are currently attempting to set up the same model for allergen-induced LPRs.

Such models should allow the study of T cell dependent cutaneous reactions in a human environment. They should provide critical insights into the understanding of some immunological disorders such as allergy and might be useful to test new therapeutic targets.

CONCLUSION

Acknowledgements

The authors warmly acknowledge all the members of Barry Kay's laboratory, at the National Heart and Lung Institute in London, where a part of this work was performed, as well as Qutayba Hamid, Meakins Christie Laboratories in Montreal, who was a main contributor to this work.

REFERENCES

1. Coombs RRA, Gell PGH. Classification of allergic reactions responsible for clinical hypersensitivity and disease. In: Coombs RRA, Gell PGH, eds. Clinical aspects of immunology. Oxford: Blackwell Scientific publications, 1968: 575.

2. DeShazo RD, Levinson AI, Dvorak HF. The late phase skin reaction: paradigm or epiphenomena ? Ann Allergy 1983; 51: 166-72.

3. Kaplan G, Nusrat A, Witmer MD, Nath I, Cohn ZA. Distribution and turnover of Langerhans cells during delayed immune responses in human skin. J Exp Med 1987; 165: 763-76.

4. Grabbe S, Steinbrink K, Steinert M, Luger TA, Schwarz T. Removal of the majority of epidermal Langerhans cells by topical or systemic steroid application enhances the effector phase of murine contact hypersensitivity. J Immunol 1995; 155: 4207-17.

5. Gaga M, Frew AJ, Varney V, Kay AB. Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase reactions and classical delayed-type hypersensitivity in man. J Immunol 1991; 147: 816-22.

6. Scheynius A, Klareskog L, Forsum U. In situ identification of T lymphocyte subsets and HLA-DR expressing cells in the human skin tuberculin reaction. Clin Exp Immunol 1982; 49: 325-30.

7. Bergholtz BO, Thorsby E. HLA-D restriction of the macrophage-dependent response of immune human T lymphocytes to PPD in vitro: inhibition by anti-HLA-DR antisera. Scand J Immunol 1978; 8: 63-73.

8. Vyakarnam A, Lachmann PJ. Migration inhibition factor secreting human T-cell lines reactive to PPD: a study of their antigen specificity, MHC restriction and the use of Epstein-Barr virus transformed B cell lines as requirement for antigen-presenting cells. Immunology 1984; 53: 601-10.

9. Gocinski BL, Tigelaar RE. Roles of CD4⁺ and CD8⁺ T cells in murine contact hypersensitivity revealed by in vivo monoclonal antibody depletion. J Immunol 1990; 144: 4121-8.

10. Viraben R, Aquilana C, Cambon L, Bazex J. Allergic contact dermatitis in HIV-positive patients. Contact Dermatitis 1994; 31: 326-7.

11. Suzumura Y, Ohasi M. Immunoelectron microscopic localization of HLA-DR antigen on mast cells and vessels in normal and tuberculin-reactive skin. Am J Dermatopathol 1991; 13: 568-74.

12. Love KS, Lakshmanan RR, Butterfield JH, Fox CC. IFN-gamma-stimulated enhancement of MHC class II antigen expression by the human mast cell line HMC-1. Cell Immunol 1996; 170: 85-90.

13. Fox CC, Jewell SD, Whitacre CC. Rat peritoneal mast cells present antigen to a PPD-specific T cell line. Cell Immunol 1994; 158: 253-64.

14. Marchal G, Seman M, Milon G, Truffa-Bachi P, Zilberfarb V. Local adoptive transfer of skin delayed-type hypersensitivity initiated by a single T lymphocyte. J Immunol 1982; 129: 954-8.

15. Lombardi G, Piccolella E, Vismara D, Colizzi V, Asherson GL. Candida albicans polysaccharide extract (MPPS) and PPD stimulate the productionn of interleukin-1 and lymphocyte proliferation. Clin Exp Immunol 1984; 58: 581-6.

16. Tsicopoulos A, Hamid Q, Varney V, Ying S, Moqbel R, Durham SR, Kay AK. Preferential messenger RNA expression of Th1-type cells (IFN-gamma⁺, IL-2⁺), in classical delayed-type (tuberculin) hypersensitivity reactions in human skin. J Immunol 1992; 148: 2058-61.

17. Wangoo A, Cook HT, Taylor GM, Shaw RJ. Enhanced expression of type 1 procollagen and transforming growth factor-beta in tuberculin-induced delayed type hypersensitivity. J Clin Pathol 1995; 48: 339-45.

18 Ohmen JD, Hanifin JM, Nickoloff BJ, Rea TH, Wyzykowski R, Kim J, Jullien D, McHugh T, Nassif AS, Chan SC, Modlin RL. Overexpression of
IL-10 in atopic dermatitis. Contrasting cytokine patterns with delayed-type hypersensitivity reactions. J Immunol 1995; 154: 1956-63.

19. Robinson DS, Tsicopoulos A, Meng Q, Durham SR, Kay AB, Hamid Q. Increased interleukin-10 messenger RNA expression in atopic allergy and asthma. Am J Respir Cell Mol Biol 1996; 14: 113-7.

20. Li L, Elliott JF, Mosmann TR. IL-10 inhibits cytokine production, vascular leakage, and swelling during T helper 1 cell-induced delayed-type hypersensitivity. J Immunol 1994; 153: 3967-78.

21. Kaplan G, Luster AD, Hancock G, Cohn ZA. The expression of a gamma interferon-induced protein (IP-10) in delayed immune responses in human skin. J Exp Med 1987; 166: 1098-108.

22. Bonecchi R, Bianchi G, Bordignon PP, D'Ambrosio D, Lang R, Borsatti A, Sozzani S, Allavena P, Gray PA, Mantovani A, Sinigaglia F. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J Exp Med 1998; 187: 129-34.

23. Devergne O, Marfaing-Koda A, Schall TJ, Leger-Ravet MB, Sadick M, Peuchmaur M, Crevon MC, Kim KJ, Galanaud P, Emilie D. Production of the RANTES chemokine in delayed-type hypersensitivity reactions: involvement of macrophages and endothelial cells. J Exp Med 1994; 179: 1689-94.

24. Larsen CG, Thomsen MK, Gesser B, Thomsen PD, Deleuran BW,
Nowak J, Skodt V, Thomsen HK, Deleuran M, Thestrup-Pedersen K, Harada A, Matsushima K, Menné T. The delayed-type hypersensitivity reaction is dependent on IL-8. Inhibition of a tuberculin skin reaction by an anti-IL-8 monoclonal antibody. J Immunol 1995; 155: 2151-7.

25. Rand ML, Warren JS, Mansour MK, Newman W, Ringler DJ. Inhibition of T cell recruitment and cutaneous delayed-type hypersensitivity-induced inflammation with antibodies to monocyte chemoattractant protein-1. Am J Pathol 1996; 148: 855-64.

26. Boring L, Gosling J, Chensue SW, Kunkel SL, Farese RV, Broxmeyer HE, Charo IF. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice. J Clin Invest 1997; 100: 2552-61.

27. Norris P, Poston RN, Thaoms DS, Thornhill M, Hawk J, Haskard DO. The expression of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in experimental cutaneous inflammation: a comparison of ultraviolet B erythema and delayed hypesensitivity. J Invest Dermatol 1991; 96: 763-70.

28. State ND, Justen JM, Sly LM, Beaudet AL, Bullard DC. Inhibition of delayed-type contact hypersensitivity in mice deficient in both E-selectin and P-selectin. Blood 1996; 88: 2973-9.

29. Tipping PG, Huang XR, Berndt MC, Holdsworth SR. P-selectin directs T lymphocyte-mediated injury in delayed-type hypersensitivity responses: studies in glomerulonephritis and cutaneous delayed-type hypersensitivity. Eur J Immunol 1996; 26: 454-60.

30. Austrup F, Vestweber D, Borges E, Lohning M, Brauer R, Herz U, Renz H, Hallmann R, Schefffold A, Radbruch A, Hamann A. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. Nature 1997; 385: 81-3.

31. Blackley CH. Experimental researches on the causes and nature of catarrhus aestivus (hay fever or hay asthma). London: Tindall and Cox, 1873: 77.

32. Dolovich J, Hargreave FE, Chalmers R, Shier KJ, Gauldie J, Bienenstock J. Late allergic cutaneous responses in isolated IgE-dependent reactions. J Allergy Clin Immunol 1973; 52: 38-46.

33. Talbot SF, Atkins PC, Valenzano M, Zweiman B. Correlations of in vivo mediator release with late cutaneous allergic responses in humans. J Allergy Clin Immunol 1984; 74: 819-26.

34. Pienkowski MM, Adkinson NF, Plaut M, Norman PS, Lichtenstein LM. Prostaglandin D2 and histamine during the immediate and the late-phase components of allergic cutaneous responses. J Allergy Clin Immunol 1988; 82: 95-100.

35. Atkins PC, Schwartz LB, Adkinson NF, Von Allmen C, Valenzano M, Zweiman B. In vivo aAntigen-induced cutaneous mediator release: simultaneous comparisons of histamine, tryptase and prostaglandin D2 release and the effect of oral corticosteroid administration. J Allergy Clin Immunol 1990; 86: 360-70.

36. Massey W, Friedman B, Kato M, Cooper P, Kagey-Sobotka A, Lichtenstein LM, Schleimer RP. Appearance of granulocyte-macrophage colony-stimulating factor activity at allergen-challenged cutaneous late-phase reaction sites. J Immunol 1993; 150: 1084-92.

37. Hogaboam C, Kunkel SL, Strieter RM, Taub DD, Lincoln P, Standiford TJ, Lukacs NW. Novel role of transmembrane SCF for mast cell activation and eotaxin production in mast cell-fibroblast interactions. J Immunol 1998; 160: 6166-71.

38. Yano K, Yamagushi M, de Mora F, Lantz CS, Butterfield JH, Costa JJ, Galli SJ. Production of macrophage inflammatory protein-1 alpha by human mast cells: increased anti-IgE-dependent secretion after IgE-dependent enhancement of mast cell IgE binding ability. Lab Invest 1997; 77: 185-93.

39. Rajakulasingam K, Hamid Q, O'Brian F, Shotman E, Jose PJ, Williams TJ, Jacobson M, Barkans J, Durham SR. RANTES in human allergen-induced rhinitis: cellular source and relation to tissue eosinophilia. Am J Respir Crit Care Med 1997; 155: 696-703.

40. Ying S, Taborda-Barata L, Meng Q, Grant JA, Barkans J, Durham SR, Kay AB. High-affinity immunoglobulin E receptor (Fc epsilon RI)-bearing eosinophils, mast cells, macrophages and Langerhans cells in allergen-induced late cutoeneous reactions in atopic subjects. Immunology 1998; 93: 281-8.

41. Frew AJ, Kay AB. The relationship between CD4⁺ lymphocytes, activated eosinophils and the magnitude of the allergen-induced late phase skin reaction in man. J Immunol 1988; 141: 4158-64.

42. Kon OM, Sihra BS, Compton CH, Leonard TB, Kay AB, Barnes NC. Randomised dose ranging, placebo-controlled study of chimeric antibody to CD4 (keliximab) in chronic severe asthma. Lancet 1998; 352: 1109-13.

43. Yssel H, Johnson KE, Schneider PV, Wideman J, Terr A, Kastelein R, De Vries JE. T cell activation-inducing epitopes of the house dust mite allergen Der pI. Proliferation and lymphokine production patterns by DER pI-specific CD4⁺ T cell clones. J Immunol 1992; 148: 738-45.

44. Dudler T, Altmann F, Carballido JM, Blaser K. Carbohydrate-dependent, HLA class II-restricted, human T cell response to the bee venom allergen phospholipase A2 in allergic patients. Eur J Immunol 1995; 25: 538-42.

45. Kay AB, Ying S, Varney V, Durham SR, Moqbel R, Wardlaw AJ,
Hamid Q. Messenger RNA expression of the cytokine gene cluster, IL-3,
IL-4, IL-5, and GM-CSF in allergen-induced late-phase reactions in atopic subjects. J Exp Med 1991; 173: 775-8.

46. Ying S, Taborda-Barata L, Meng Q, Humbert M, Kay AB. The kinetics of allergen-induced transcription of messenger RNA for monocyte chemotactic protein-3 and RANTES in the skin of human atopic subjects: relationship to eosinophil, T cell, and macrophage recruitment. J Exp Med 1995; 181: 2153-9.

47. Zweiman B, Kaplan AP, Tong L, Moskowitz AR. Cytokine levels in inflammatory responses in developing late-phase allergic reactions in the skin. J Allergy Clin Immunol 1997; 100: 104-9.

48. Lamkhioued B, Renzi PM, Abi-Younes S, Garcia-Zepada EA, Allakhverdig Z, Ghaffar O, Rothenberg MD, Luster AD, Hamid Q. Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation. J Immunol 1997; 159: 4593-601.

49. Ying S, Robinson DS, Meng Q, Rottman J, Ringler DJ, Mackay CR, Daugherty BL, Springer MS, Durham SR, Williams TJ, Kay AB. Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma. Association with airway hyperresponsiveness and predominant co-localization of eotaxin mRNA to bronchial and endothelial cells. Eur J Immunol 1997; 27: 3507-16.

50. Sallusto F, Mackay CR, Lanzavecchia A. Selective expression of the eotaxin receptor by human T helper 2 cells. Science 1997; 277: 2005-7.

51. Leung DYM, Pober JS, Cotran RS. Expression of endothelium-leukocyte adhesion molecule-1 in elicited late phase allergic reactions. J Clin Invest 1991; 87: 1805-9.

52. Tsicopoulos A, Hamid Q, Haczku A, Jacobson M, Durham SR, North J, Barkans J, Corrigan C, Meng Q, Moqbel R, Kay AB. Kinetics of cell infiltration and cytokine mRNA expression after intradermal challenge with allergen and tuberculin in the same atopic individuals. J Allergy Clin Immunol 1994; 94: 764-72.

53. Shirakawa T, Enomoto T, Shimazy S, Hopkin JM. The inverse association between tubeculin responses and atopic disorder. Science 1997; 275: 77-9.

54. Erb KJ, Holloway JW, Sobeck A, Moll H, Le Gros G. Infection with mice with mycobacterium bovis-Bacillus Calmette Guerin (BCG) suppresses allergen-induced airway eosinophilia. J Exp Med 1998; 187: 561-9.

55. Varney VA, Hamid QA, Gaga M, Sun Ying, Jacobson M, Frew AJ, Kay AB, Durham SR. Influence of grass pollen immunotherapy on cellular infiltration and cytokine mRNA expression during allergen-induced late-phase cutaneous responses. J Clin Invest 1993; 92: 644-51.

56. Akoum H, Tsicopous A, Vorng H, Wallaert B, Dessaint JP, Joseph M, Hamid Q, Tonnel AB. Venom immunotherapy modulates IL-4 and IFN-gamma m RNA expression of peripheral T lymphocytes. Immunology 1996; 87: 593-8.

57. Jutel M, Pichler WJ, Skrbic D, Urwyler A, Dahinden C, Müller UR. Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in specific allergen-stimulated T cell cultures. J Immunol 1995; 154: 4194-7.

58. O'Hehir RE, Yssel H, Verma S, De Vries JE, Spits H, Lamb JR. Clonal analysis of differential lymphokine production in peptide and superantigen induced T cell anergy. Int Immunol 1991; 3: 819-26.

59. Tsicopoulos A, Pestel J, Fahy O, Vorng H, Vandenbusche F, Porte H, Eraldi L, Wurtz A, Akoum H, Hamid Q, Wallaert B, Tonnel AB. Tuberculin-induced delayed type hypersensitivity in a model of hu-PBMC-SCID mice grafted with autologous skin. Am J Pathol 1998; 152: 1681-8.