Anti-Hapten Antibodies in Response to Skin Sensitization

Contact Dermatitis • Review Article COD Contact Dermatitis

Anti-hapten antibodies in response to skin sensitization

Helen Singleton1, Amy Popple1, Nichola Gellatly2, Gavin Maxwell2, Jason Williams3, Peter S. Friedmann4, Ian Kimber1 and Rebecca J. Dearman1 1Department of Toxicology, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK, 2Unilever Safety and Environmental Assurance Centre, Colworth Science Park, Bedford, MK44 1LQ, UK, 3Salford Royal NHS Foundation Trust, Salford, M6 8HD, UK, and 4Division of Infection, Inﬂammation & Immunity, University of Southampton, Southampton, SO17 1BJ, UK doi:10.1111/cod.12486

Summary Whereas T lymphocyte (T cell) activation is the key event in the acquisition of skin sensitization and subsequent elicitation of allergic contact dermatitis, the humoral component of immune responses to organic contact allergens has received little consideration. There is evidence that, in experimental animals, topical exposure to potent contact allergens is associated with B cell activation and proliferation, and hapten-specific antibody production. However, there is very limited evidence available for anti-hapten antibody responses being induced following topical exposure of humans to contact allergens. Nevertheless, it is important to appreciate that there are almost no negative studies in which evidence for antibody production as the result of skin sensitization has been sought and not found. That is, there is absence of evidence rather than evidence of absence. Furthermore, exposure to chemical respiratory allergens, in which the skin has been implicated as a potential route of sensitization, results in anti-hapten antibody responses. It is proposed that skin sensitization to contact allergens will normally be accompanied by antibody production. The phenomenon is worthy of investigation, as anti-hapten antibodies could potentially influence and/or regulate the induction of skin sensitization. Moreover, such antibodies may provide an informative correlate of the extent to which sensitization has been acquired.

Key words: allergic contact dermatitis; antibody response; B cell; hapten; IgG; IgM; skin sensitization.

From an immunological perspective, the acquisition of the roles played by T cells in skin sensitization and allergic skin sensitization and the elicitation of allergic contact contact dermatitis (4–7). dermatitis are immune and inflammatory responses However, at least in animals, contact allergens provoke mediated by antigen (hapten)-specific T cells (1–3). For both humoral and cell-mediated immune responses. this reason, it is the activation, differentiation and regula- Thus, topical encounter with a contact allergen will tion of T cells upon which attention has been focused, and induce anti-hapten antibody formation in addition to the there are available excellent recent reviews that describe activation and expansion of responsive T cells (8–10). Given the predominant role played by T cells in skin sensitization, it is perhaps not surprising that anti-hapten antibody responses have received comparatively little Correspondence: Dr Helen Singleton, Faculty of Life Sciences, University of attention. This is unfortunate because, in theory, a Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK. Tel: +44 1612755368. E-mail: [email protected] hapten-specific antibody has the ability to influence both the acquisition of skin sensitization and the subsequent Conﬂicts of interest: The authors report no conﬂicts of interest. The authors elicitation of allergic reactions. Moreover, it is possible alone are responsible for the content and writing of this article. that the vigour and isotype distribution of anti-hapten Accepted for publication 21 September 2015 antibodies induced by exposure to contact allergens

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Contact Dermatitis 1 ANTI-HAPTEN ANTIBODIES IN RESPONSE TO SKIN SENSITIZATION • SINGLETON ET AL. may provide correlates of the quality of T lymphocyte contact allergens will induce antibody responses, in addi- responses and the extent of skin sensitization. tion to T lymphocyte responses. This should come as Here, we review the available evidence from experi- no real surprise, because the vast majority of immuno- mental animals and humans that topical exposure to con- gens are known to stimulate both T cell and antibody tact allergens will elicit antibody responses. We consider responses. also whether such antibodies have the ability to influence the development of sensitization and/or the elicitation of allergic contact dermatitis. Anti-hapten antibody responses induced following exposure to contact allergens in experimental animals T cell responses in allergic contact dermatitis Consistent with the ability of Th1 cells to support anti- Although a detailed review of T cell responses in allergic body responses, there is clear evidence from studies contact dermatitis is outside the scope of this article, it is conducted in experimental animals that detectable necessary to briefly outline what is known of the role ofT antibody production is induced following exposure cells in the acquisition of skin sensitization. It is generally to skin-sensitizing chemicals. Very early work in the accepted that, in humans and in relevant experimental 1940s using guinea-pigs showed that skin hypersensi- rodent models of skin sensitization, both hapten-specific tivity reactions to the known human contact allergen CD8+ T cytotoxic (Tc) and CD4+ T helper (Th) cell sub- 2,4-dinitrochlorobenzene (DNCB) could be transferred sets play important roles (11, 12). Tc and Th cell pop- with immune serum to naive guinea-pigs, indicating a ulations have been shown to polarize into characteris- humoral (antibody) component (25). However, in these tic subsets with distinctive cytokine expression profiles experiments, animals were immunized by repeated intra- and functions. The most well studied of these subsets are dermal injection of free chemical, which is not directly type 1 (Tc1 and Th1) cells, which express interferon-𝛾, relevant to the human experience. Some decades later, and type 2 (Tc2 and Th2) cells, which preferentially it was shown that topical treatment of guinea-pigs with secrete interleukin (IL)-4, IL-5, and IL-13 (13–15). Thus, DNCB resulted in a marked increase in the number of following challenge of sensitized individuals with aller- germinal centres in skin-draining lymph nodes, indica- gen, there is rapid recruitment of skin-homing Tc1 cells tive of B cell activation and the production of antibodies that cause apoptosis of keratinocytes, and CD4+ Th1 (26). Furthermore, topical application to mice of the cells that release proinflammatory cytokines, activating experimental skin sensitizer oxazolone or picryl chloride keratinocytes, and other skin cells, contributing to the [trinitrochlorobenzene (TNCB)] was shown to stimulate inflammatory reaction (2, 7, 11). Other subsets ofCD4+ antibody production, detected as a function of the ability T cells with regulatory properties appear to restrict the of serum to agglutinate haptenated red blood cells (RBCs) magnitude of the response to hapten, particularly potent (27, 28), although this effect was somewhat mouse sensitizers (2, 16–18). Whereas Th1 cells are associated strain-dependent (28). However, interpretation of the with cell-mediated reactions such as allergic contact der- latter results was hampered by the detection of antibodies matitis, the primary function of Th2 cells is to help B in non-immune sera, suggesting that there was consider- lymphocytes (B cells) make antibodies (19, 20). How- able background in the assay, or that natural antibodies ever, Th1 cells can also provide help for B cells and anti- are present that are reactive with some haptens (29–31). body production; thus, experiments with protein antigens More sophisticated techniques for antibody detec- conducted in mice showed that Th1 and Th2 cells could tion, particularly enzyme-linked immunosorbent assays support B cell responses to a similar extent in vivo (21), (ELISAs), have permitted the improved characterization with both subsets sustaining IgM production, whereas of anti-hapten antibody responses, and have provided Th1 and Th2 cells preferentially induced IgG2a and IgG1 a more robust body of evidence that such antibodies responses, respectively (22). This phenomenon is paral- are produced following skin sensitization. These studies leled by human T cell subsets, with the IgG1 subclass encompass a relatively narrow range of skin sensitizers, being driven by Th1 cells and the IgG4 subclass being being confined to the experimental contact allergens associated with Th2-dominant responses (23, 24). oxazolone and TNCB, and the human allergen DNCB and The fact that Th1 cells, which constitute one of the the chemically related dinitrofluorobenzene (DNFB). A main T cell subpopulations activated in contact sensi- number of reports have shown that specific IgM produc- tization, can provide B cell help supports the assertion tion is induced in mice treated topically with oxazolone that antibodies may be produced in individuals with con- or TNCB (9, 32–34). This antibody class can be detected tact allergy. There is reason to assume, therefore, that within 24 hr of sensitization with hapten (32, 33). There

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 2 Contact Dermatitis ANTI-HAPTEN ANTIBODIES IN RESPONSE TO SKIN SENSITIZATION • SINGLETON ET AL. is also good evidence that simple epicutaneous exposure Anti-hapten antibody responses induced of rodents to contact allergens can induce the production following exposure to contact allergens of hapten-specific IgG antibodies. Thus, topical appli- in humans cation of DNCB or DNFB induces relatively vigorous There is limited evidence available for anti-hapten anti- IgG responses in mice, and somewhat more variable body responses being detected following topical exposure responses in rats (10, 35–40). In mice, anti-DNCB IgA of humans to chemical contact allergens. That is, there has also been detected (10), and kinetics studies have are very few studies that have reported analysis of shown that measurable levels of IgG are not observed antibody responses following contact sensitization; until some 8 days after the initiation of exposure (10, rather, there are many negative studies. An early study, 36), or after repeat exposure to allergen some 10 days reported in 1969, investigated the presence of anti-hapten later (38). Consistent with the early reports of haemag- antibodies in actively sensitized human volunteers hospi- glutinating antibodies (27, 28), skin sensitization with talized for diverse diseases unrelated to immune function oxazolone also induces a hapten-specific IgG response (56). Here, 17 volunteers were contact-sensitized by open in mice, as measured by ELISA (37, 41–43). In those topical application of TNCB, and a further 8 patients murine studies where the isotype of the IgG response were sensitized to DNFB, with between four and six has been characterized, detectable anti-hapten IgG1, biweekly treatments with the same chemical. All individ- IgG2a and IgG2b production has been recorded (10, uals developed delayed-type hypersensitivity responses 44), with several reports of a preferential IgG2a response in the skin, but, in serum derived from serial (weekly) to both DNCB and oxazolone (36, 41, 45), consistent bleeds, there was no detectable antibody, measured as with selective Th1 cell activation. Interestingly, there a function of haemagglutinating activity. In parallel are also reports that IgE may also be detected following experiments, immunization with TNCB conjugated with topical exposure of mice to contact allergens, particu- human serum albumin carrier protein by subcutaneous larly when there is repeated skin exposure and through injection resulted in delayed-type hypersensitivity reac- abraded or damaged skin (46, 47). In general, however, it tions within 7 days of immunization, and the production appears that, when less vigorous exposure protocols are of agglutinating antibodies within 2 weeks of exposure. used, contact allergens are not associated with increases This antibody response was relatively transient, peaking in serum IgE, usually measured as a function of the after 3–4 weeks and, in the absence of further antigen increase in the total serum concentration of this isotype stimulation, being undetectable after 90–110 days. It (48–51). should be noted that these analyses were conducted Further evidence for B cell activation and proliferation prior to the development of more sensitive and sophis- in murine models of contact sensitization comes from ticated techniques such as ELISAs to measure specific examination of alternative endpoints to antibody produc- antibody production, and that the same authors found tion. Autoradiographic analyses of oxazolone-activated that very high doses of contact allergen were required to lymph node tissue showed an increased frequency stimulate detectable anti-hapten antibody production in of proliferating cells in the lymphoid follicles (B cell guinea-pigs with the same techniques (56). areas) (37). More sophisticated flow cytometric analyses Since that time, few studies have measured antibody using a number of skin sensitizers [DNCB, hexyl cinna- production following exposure to contact allergens. There mal (𝛼-hexyl cinnamaldehyde), TNCB, isoeugenol, and are some reports of antibody responses to selected contact eugenol] showed that epicutaneous application of these allergens; however, these have been observed following chemicals to mice, unlike application of skin irritants, exposure via non-topical routes. For example, following was associated with increases in the number of B220+ long-term exposure to contact-sensitizing metals such as lymphocytes (B cells) and their activation status, mea- nickel and cobalt through metallic orthopaedic devices, a sured as a function of upregulation of expression of the marked increase in metal-specific antibodies was detected costimulatory marker CD86 and major histocompatibil- (57). Thus, in the 10 patients studied, pre-implant and ity complex class II, 5 days after the initiation of exposure post-implant serum analyses showed that the majority (52–55). showed an increase in the level of antibody (IgG, IgM, All together, there is very convincing evidence that, in IgA, or IgE) directed against at least one metal–protein experimental animals (primarily rodents), topical expo- carrier complex. Similarly, IgG against the contact aller- sure to a number of contact sensitizers is associated with gen formaldehyde has been detected, but only in individ- B cell activation and proliferation and the production of uals who have been exposed to this material either intra- hapten-specific antibodies, particularly of the IgM and IgG venously (through sterilization of medical devices) or by subtypes. inhalation (occupationally as arc welders) (58, 59). In

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Contact Dermatitis 3 ANTI-HAPTEN ANTIBODIES IN RESPONSE TO SKIN SENSITIZATION • SINGLETON ET AL. both cases, anti-formaldehyde IgG was detected in some respiratory allergy being confined to the lung, there is of the individuals (6/19 and 5/6, respectively), but it was an increasing consideration that skin exposure may well of relatively low titre. be an important route in the induction and subsequent Intriguingly,Vojdani et al. recently described the detec- acquisition of respiratory sensitization (72–76). There- tion of both hapten-specific IgG and IgM against a number fore, these observations represent a precedent for the of chemicals in a proportion of serum samples derived ability of the skin to provide a route for the development from 400 healthy individuals (60). Although information of antibody responses. regarding the amount and route of chemical exposure is unavailable, hapten-specific antibody responses to some contact allergens, including formaldehyde, parabens, Technical difﬁculties in detecting anti-hapten bisphenol-A, and dinitrophenyl (DNP) (the leaving group antibody responses of the haptens DNCB and DNFB) have been reported. Although it seems likely that the paucity of studies exam- It is of interest that much earlier studies also reported ining antibody responses to contact allergens is at least in anti-DNP IgM and IgG activity in human serum drawn part attributable to the fact that the appropriate experi- from non-sensitized donors, with the suggestion that ments have not been conducted, it is important to empha- these antibodies may represent ‘natural’ antibodies or size that there are technical difficulties in measuring antibodies that are cross-reactive with a number of dif- anti-hapten serum antibody responses in humans. The ferent antigens (29–31), and with some evidence of nature of the hapten–protein conjugates that represent association with bacterial infection (61). However, this the most relevant form for detecting antibodies remains does not explain the findings of antibody responses to the uncertain, and, in human studies, it is often difficult to other haptens described by Vojdani et al. (60). control the timing of serum isolation relative to the last In considering the evidence that contact allergens exposure (77, 78). In addition, studies in mice using induce anti-hapten antibody responses in humans, the crude reagents (haptenated RBCs) have suggested that the data relating to metal allergens must be treated with some anti-hapten antibodies produced following topical expo- caution. Whether metals are able to provoke humoral sure are of relatively low affinity and require highly sub- immune responses is uncertain, and, in those reports stituted substrates for detection (27). in which anti-metal antibodies have been described, the specificity of the analytical methods is not known. Although the evidence is lacking with respect to detec- A role for anti-hapten antibodies and B cells tion of antibodies following topical exposure of humans in the induction and regulation of skin to chemical contact allergens, there is very clear evidence sensitization that exposure to other types of chemical hapten does Given that there is reason to believe that the elicitation of indeed provoke strong antibody responses. Thus, expo- hapten-specific antibodies will result from topical exposure to chemicals that cause respiratory sensitization, sure to contact allergens, at least in experimental animals, such as the acid anhydrides and the diisocyanates, is the potential significance of such antibodies and/or Bcell often associated with the development of specific IgG, activation for the development of contact sensitization and, considerably less frequently, the production of should be acknowledged. Necessarily, the experiments specific IgE (62–67). There are many commonalities that support a role for antibody and B cells in the acquisi- between chemical contact and respiratory allergens, as tion of skin sensitization have been conducted exclusively both types of allergen are: protein-reactive or metabo- in rodents, and there have been sporadic reports that lized to a protein-reactive species, form hapten–protein antibodies may play a variety of roles in the induction conjugates that are recognized by the immune system, of skin sensitization or elicitation of allergic contact der- induce the expression of proinflammatory cytokines, matitis. Thus, in gene-targeted JH−/− mice, in which the and are immunogenic (68–71). The differences between disruption in IgH rearrangement specifically results in the these classes of chemical allergen apparently reside in the absence of B cells, impaired challenge-induced increases quality of the immune response that is stimulated and the in ear thickness were observed in TNCB-sensitized route of elicitation, namely Th1/Tc1 for contact sensitiz- mice, suggesting that B cells are necessary for optimal ers, and Th2 for chemical respiratory sensitizers (69, 70), contact sensitization in mice (33). It was shown that which results in the divergent clinical manifestations of TNCB-specific IgM was detected in the serum within delayed-type hypersensitivity skin reactions, and asthma 1 day of immunization, and it was hypothesized that and rhinitis, respectively. One further commonality is this antibody was important in the local recruitment of that, despite the clinical manifestations of chemical effector T cells (32, 33), through mast cell and platelet

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 4 Contact Dermatitis ANTI-HAPTEN ANTIBODIES IN RESPONSE TO SKIN SENSITIZATION • SINGLETON ET AL. activation, resulting in increased vascular permeability elicitation of an antibody response, the priority being via release of serotonin and tumour necrosis factor-𝛼 (79, cell-mediated immunity. However, it is well established 80). Indeed, the differential sensitivity of BALB/c strain that skin exposure of mice to proteins will elicit an anti- mice and C57BL/6 strain mice to oxazolone-induced body response (90), and it is now postulated that skin contact sensitization was suggested to be atributable to exposure to peanut proteins, and possibly other food increased numbers of IgM-producing B cells and higher allergens, can induce IgE-mediated allergic sensitiza- levels of hapten-specific IgM in the more sensitive BALB/c tion (91). Furthermore, there is a growing appreciation strain mouse (8). that sensitization of the respiratory tract to chemical There have also been some reports that B cells may play respiratory allergens, associated with IgG and, in some a regulatory role, serving to constrain elicitation reac- cases, IgE responses can be acquired via skin contact tions. For instance, Katz et al. described B lymphocyte (72–76). suppressor cells in guinea-pigs that act to reduce elicita- If, on the basis of the above arguments, it is accepted tion reactions (81). Furthermore, polyclonal B cell activa- that antibody production is a common, if not invariable, tors, such as lipopolysaccharide, inhibited contact sensi- consequence of the development of skin sensitization to tivity to oxazolone in mice when administered 24 hr prior organic contact allergens (as indicated above, metal aller- to sensitization (82). There are also other, more recent, gens possibly representing a special case), then it is appro- reports. Thus, in mice depleted of B cells 7 days prior to priate to consider what the relevance and potential utility or 2 days after sensitization with oxazolone, enhanced of this may be. challenge-induced ear-swelling responses were recorded It could be argued that the simultaneous, or nearly (83). Similar results were observed in CD19-deficient mice simultaneous, induction of an antibody response during with the chemically unrelated hapten DNFB, indicating the acquisition of T cell-mediated skin sensitization may that it is this B cell-specific surface molecule that is impor- have some immunoregulatory consequences. That is, the tant in this regulatory activity.In these mice, delayed-type presence of hapten-specific antibody could, in theory,aug- hypersensitivity reactions were exacerbated as compared ment or downregulate specific T cell responses to the same with wild-type controls, with respect to both the dura- allergen. This possibility is supported by a variety of obser- tion of the response and the degree of inflammation (84). vations derived from animal studies. However, the possible Other examples of potential regulatory B cell function in regulatory role of antibodies in influencing the develop- experimental (mouse) models of contact hypersensitivity ment or vigour of skin sensitization has not been investi- have also been described (85, 86). gated in a systematic way in experimental animals, and not at all in humans. One interesting speculation is that antibodies may serve to remove or mask haptens and Discussion thereby inhibit or limit T cell responses, but this, to our Taken together, the data reviewed here suggest that chem- knowledge, has not been addressed experimentally. ical allergens have a clear potential to induce the produc- Even if the possible immunological and biological con- tion of hapten-specific antibodies, the exceptions to this sequences of anti-hapten antibodies are unproven with rule probably being metal contact allergens. The argu- regard to skin sensitization, they may have some poten- ment is that the lack of evidence for detectable anti- tial utility in toxicological considerations of allergic con- body responses in humans sensitized to contact allergens tact dermatitis. For instance, it is now well established is attributable to there being available too few relevant that contact allergens vary significantly, and by up to investigations; that is, absence of evidence rather than five orders of magnitude, with respect to their relative evidence of absence. The ability of contact allergens to skin-sensitizing potency. Although there is no reason to elicit antibody responses should not be unexpected from suppose that antibody responses as such contribute to rel- an immunological perspective. There is no reason why ative potency, it may be that they serve as quantitative a chemical allergen, in the form of a hapten–protein biomarkers of skin sensitization. If that proved to be the complex formed in vivo, should not induce an immune case, then serum levels of hapten-specific antibody might response capable of initiating and sustaining antibody provide an interesting correlate of the extent to which sen- production. In fact, haptens linked to carrier proteins con- sitization has been acquired. stituted one of the important tools used in some of the pio- In line with the results of animal studies, it has been neering investigations that first showed the requirement shown that sensitization to a contact allergen in humans for T cell help in most antibody responses (87–89). may also be influenced by the quality of induced T cell It could be argued, of course, that, under normal responses, and, in particular, by the balance of discrete circumstances, exposure via the skin does not favour the function subpopulations of T cells (subpopulations of

Th and Tc T cells) (92). These same T cell subsets also be accompanied by the development of an antibody influence the nature and isotype distribution of anti- response, this has not yet been formally proved. body responses, and it could be that the spectrum of It is proposed that contact allergen-induced antibody antibodies produced following exposure to a contact production warrants further investigation, particularly in allergen might correlate with the extent of sensitization humans. Irrespective of whether anti-hapten antibodies achieved. influence the induction of skin sensitization, such The conclusion drawn is that, although some evi- antibody responses could provide informative correlates dence is available to suggest that the acquisition of skin of the quality and vigour of the cell-mediated immune sensitization to organic contact allergens will normally responses induced by contact allergens.

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