IgG4 antibody assays in allergy diagnosis

Res. Immunol. 1998, 149, 263-266

© INSTITUTPASTEUR]ELsEVIER

Paris 1998

IgG4 antibody assays in allergy diagnosis R.C. Aalberse, J. Schuurman, R. van Ree and S. Stapel CLB and Laboratory for Clinical and Experimental Immunology, Academic Medical Centre, University of Amsterdam, Amsterdam

By definition, atopic allergy has been associated with IgE responses. However, the IgE system is not the only one activated in this condition. The T-cell system is involved, because IgE production is strongly if not absolutely dependent on T-cell help. These activated T cells promote the synthesis not only of IgE antibodies, but also of other isotypes, i.e. IgG1 and IgG4 antibodies. The emphasis of this presentation will be on IgG4, because this isotype is most strongly associated with atopy. However, the potential contribution of other isotypes, particularly IgG1, should not be overlooked: prior to allergen-specific immunotherapy, the absolute amount of IgG1 antibody is usually (at least for pollen and mite allergens) not very different from that of IgG4. Only after prolonged immunotherapy does IgG4 become quantitatively dominating. For reviews on IgG4, see e.g. [1-3, 14-16].

To what extent are IgG(4) antibody levels suitable diagnostic markers to indicate the level of protection of sensitized patients against allergic challenge ? Is there evidence that IgG(4) antibodies are actually protective and if so, what is their mechanism of action ?

To what extent are IgG(4) antibody levels suitable diagnostic markers indicating allergic sensitization ?

An important consideration, particularly in relation to the latter two issues, is the exact specificity of the IgG(4) antibodies. It should be kept in m i n d that most allergy tests use allergen extracts rather than single allergenic proteins, so IgG antibodies may be directed to antigens distinct from the IgE-binding allergens. The use of extracts labelled as "standardized" does not reduce the problem: these are still complex mixtures containing many unidentified components in addition to a mixture of allergens. This complex nature of allergen extracts is particularly problematic in IgG1 antibody assays. The problem becomes even greater after injections with a similar allergen extract, because then the highmolecular-weight antigens may easily contribute, whereas antigens with a molecular mass of more than 100 kDa will efficiently be excluded by the mucosal barrier.

Is there evidence that IgG(4) antibodies are actually pathogenic and if so, what is their mechanism of action ?

Moreover, even if purified allergens are used for testing, the IgE and IgG(4) antibodies are often found to be directed to distinct epitopes.

Four aspects of this IgG response will be discussed, two dealing with pragmatic serodiagnostic issues and two dealing with pathogenetic mechanisms.

Received April 1, 1998.

264

R.C. AALBERSE ET AL.

To what extent are IgG(4) antibody levels suitable diagnostic markers indicating allergic sensitization? The answer to this question depends on the allergen exposure level and the exposure route. For low-exposure allergens such as pollen and mites, IgG4 antibody levels are almost as diagnostic as IgE antibody levels. For high-exposure allergens, such as foods and animal danders, the prevalence of IgG4 antibodies approaches 100% (independent of allergic symptomatology). The level of IgG4 antibodies has occasionally been reported to be higher in subjects with associated symptomatology. However, the level of exposure has a strong effect on the IgG4 antibody level, so information on matching exposure in the symptomatic control group is essential (and usually difficult to obtain).

Is there evidence that IgG4 antibodies are actually pathogenic and if so, what is their mechanism of action ? In contrast to most other mammals, primate mast cells do not seem to possess a type III (activating) Fc-gamma receptor. Moreover, IgG4 antibodies do not bind well to any of the three conventional Fc-gamma receptors. Direct allergen-induced degranulation of human mast cells or basophilic leucocytes "sensitized" with human IgG antibodies is not found in the absence of complement. Two situations have been described in which IgG antibodies may cause complementindependent histamine release: IgG4 antibodies to IgE may bind to cell-bound IgE; this interaction does not result in cell activation because IgG4 behaves as a monovalent antibody. However, subsequent in vitro challenge with a cross-linking anti-IgG4 antibody will induce cell triggering. "Stripped" basophils sensitized with a single mouse/human chimaeric IgE monoclonal antibody to the mite allergen Der p2 will not release histamine when challenged with m o n o m e r i c allergen, but will when the allergen is polymerized by monoclonal chimaeric IgG1 or IgG4 to a non-overlapping epitope. In this special situation

(a monovalent IgE-allergen interaction in combination with a cross-linking IgG antibody), the IgG antibodies behave as quasi-anaphylactic antibodies. This quasi-anaphylactic reaction was found not only with human IgG1, but also with IgG4. This is surprising in view of the low crosslinking potential of polyclonal IgG4 antibodies. The explanation for the anomalous behaviour of IgG4 antibodies with respect to cross-linking of two antigens was found to be due to lability of the interaction between the heavy chains of IgG4, resulting in vivo in the exchange of half-molecules with other IgG4.

To what extent are IgG(4) antibody levels suitable diagnostic markers to indicate the level of protection of sensitized patients against allergic challenge? IgG4 antibody levels substantially increase following successful classical (allergen-specific) immunotherapy, and there is a very convincing d i f f e r e n c e in a l l e r g e n - s p e c i f i c IgG4 levels between placebo and verum in double-blind placebo-controlled trials. This is true particularly for pollen and mite allergens, whereas the situation is more complicated for venom allergens. In the latter case, it is less unusual to find substantial quantifies of venom-specific IgG4 in the serum before start of the treatment. However, in the subgroup of subjects with a low antibody IgG4 level before treatment, the increase in IgG4 antibodies is again usually very convincing. From the serodiagnostic point, the most important question is, of course: how useful is the (change in) allergenspecific IgG4 level for the evaluation of the therapy ? The answer physicians would like to hear is: there is a certain protective antibody level, anyone abow~ this IgG4 level will be doing fine, anyone below this level is likely to remain symptomatic. On a statistical basis, this may be true, but it is evidently not true for all individual patients. This is not unexpected, either for the "falsepositives" (lots of specific: IgG4, but no clear clinical improvement) or for the false-negatives (a decrease in symptoms without much change in IgG4, e.g. a patient in the placebo group). "Falsepositives" (IgG4, yet symptoms) are expected if

A B S T R A C T : N E W T H E R A P Y I N A L L E R G I C DISEASES

the spectrum of the IgG4 antibodies is different from that of the IgE antibodies, either on the individual allergen level or on the epitope level; the most obvious case is when symptoms are (partially) due to other allergen sources or to nonallergenic causes. "False-negatives" (little IgG4, fewer symptoms) are to be expected since the placebo response rate for allergic diseases is usually substantial; there is no reason to assume that a placebo response is related to protection by IgG4 antibodies.

Is there evidence that IgG(4) antibodies are a c t u a l l y p r o t e c t i v e a n d if so, w h a t is their mechanism of action? It is likely that venom immunotherapy is different from immunotherapy with inhalant allergens. Allergic reactions to venom are typically acute, whereas the main effect of immunotherapy with inhalant allergens is to reduce the late-phase reaction. Moreover, the anaphylactic response to venom is likely to be significantly affected by labile b i o l o g i c a l l y active s u b s t a n c e s in the venom, as evidenced by the patients that tolerate an injection of a commercial venom preparation and yet do react to a sting by a live insect. Part of the effect of the venom injections is the induction of "anti-toxic" immunity. This effect is most clearly seen in the decrease in the size of the toxic reaction in beekeepers during the season. The commercial venom preparations may contain the responsible "toxic" component in an toxicologically inactive, but immunogenic form, comparable to the toxoids used for routine prophylactic immunizations. While it would be preferable to directly measure these antitoxic antibodies, this is not currently possible. Therefor, in order to evaluate the response of the patient to therapy, we monitor IgG4 antibodies to some other venom component to which the patient does not already have a high antibody level before treatment; phospholipase A2 in bee venom or antigen 5 in wasp venom usually qualify, but occasionally the antibody level to these venom components is already high before treatment and then the use of another venom component is indicated. Such a use of surrogate markers is clearly a poor man's

265

choice, but is not unprecedented. Although less than ideal, it does permit an indirect way of monitoring the efficacy of the venom injections, particularly in the not uncommon situation that the d o s a g e o f the v e n o m i n j e c t i o n s c a n n o t be increased to the target level. In addition to "anti-toxic" immunity, there are indications that allergen-blocking antibodies contribute to the protective effects of allergen injections. This may be the case in venom allergy, but is more clearly so in inhalant allergy. The classical blocking antibody shields the IgE binding sites on the allergen and thus prevents allergeninduced mast cell triggering. This process can clearly be shown to operate, both in vitro as well as in vivo, particularly after preincubation of the allergen with the IgG4 antibody. The efficiency is, however, not very high, so the amounts of antibody needed for significant inhibition are high. Moreover, some of the mast cells in the airway mucosa have an intraepithelial location. In this situation, only the IgG4 in the epithelial lining fluid will be available for direct blocking of the allergen entering from the air space. There are, however, other ways in w h i c h IgG4 antibodies interfere with IgE-mediated pathology: - - IgG antibodies to epitopes that do not overlap with IgE-binding epitopes interact with the allergen; the allergen-IgG complex is fixed to the mast cell surface via allergen-specific IgE and then the allergen-bound IgG interacts with the type II Fc-gamma receptor; such an interaction has been reported to result in downregulation of mast cell activation [4-6]. In preliminary experiments with the chimaeric antibodies mentioned before, we did not find support for such downregulation, but these experiments need to be repeated and extended.

- - Another protective mechanism is linked to the effects of allergen exposure via the activation of allergen-specific T cells, with its sequelae for the late allergic reaction. Suppression of this mast-cell-dependent T-cell-mediated late inflammatory component of the allergic reaction is the most relevant effect of allergen-specific immunotherapy. This chain of events can be disrupted by IgG antibodies on at least two levels.

266

R.C. A A L B E R S E E T AL.

In both instances, there is competition between IgE and IgG for the allergen. In addition to the earlier mentioned competition between mastcell-bound IgE and IgG, there is competition between IgE bound to an antigen-presenting cell and IgG. lgE has been found to be important not only for the allergen-induced activation of mast cells, but also for the activation of allergen-specific T cells. For the latter process, IgE acts as an antigen-trapping device for antigen-presenting B cells, via the low-affinity IgE receptor (CD23) on these B cells. Via this IgE-mediated trapping mechanism, T cells have been found to become activated by much lower levels of allergen [7-13]. This process of IgE-facilitated allergen presentation is a target for competition by interfering IgG antibodies. In conclusion, IgG4 antibodies are prominently associated with allergic disease. Their role is to protect against immune damage. IgG4 protects partially because IgG4 antibody is incapable of activating effector mechanisms: it is functionally monovalent and thus does not form large complexes, it does not activate complement and it does not bind well to the three classical Fcgamma receptors. IgG4 thus protects the body against damage caused by the destructive action, particularly of IgG1 antibodies, and by the dual action of IgE antibodies: activation of mast cells and activation of allergen-specific inflammatory T cells. The measurement of IgG4 antibodies is indicated for monitoring situations with potentially dangerous chronic antigen stimulation, such as occupational exposure to organic dust or the repeated therapeutic injection of allergens. In the case of immunotherapy, the finding of an increase in allergen-specific IgG4 is a reassuring sign and the best indication currently available that the immune system has been triggered in an allergenspecific way. If no increase in antibody level is found, or the increase is largely IgG 1 rather than IgG4, this is a sign to reconsider the therapy (or the extract). If the patient does not tolerate the full dose but does show the "normal" increase in specific IgG4, the current lower dosage scheme is likely to be sufficient, but if the lower dose fails to induce an IgG4 increase, it indicates that the immune system does not receive an adequate dose of allergen.

References [1] Aalberse, R.C. (1991), IgG subclasses in atopic eczema, in : "Handbook of atopic eczema". (Ruzicka, T., Ring, J., Przybilla, B.) (pp. 166-172) Springer Verlag, Berlin Heidelberg. [2] Aalberse, R.C., Dieges, P.H., Knul-Bretlova, V., Vooren, P., Aalbers, M. & Van Leeuwen, J. (1983), IgG4 as a blocking antibody, in: "Clinical Reviews in Allergy" vol.1. (Halpern, G.M.) (pp. 289-302) Elsevier Biomedical, New York. [3] Aalberse, R.C., Van Milligen, F.J., Van 't Hof, W. & Stapel, S.O. (1993), Allergen-specific IgG4 in atopic disea:~e. Allergy, 48, 559-569. [4] Daeron, M. (1997), Negative regulation of mast cell activation by receptors for IgG. Int. Arch. Allergy IrnrnunoL, 113, 138-141. [5] Daeron, M., Latour, S., Huckel, C., Bonnerot, C. & Fridrnan, W.H. (1992), Murine Fc gamma RII and III in mast cell activation, bnmunobiology, 185, 159-174. [6] Daeron, M., Malbec, O., Latour, S., Arock, M. & Fridman, W.H. (1995), Regulation of high-affinity IgE receptor-mediated mast cell activation by murine low-affinity IgG receptors. J. Clin. Invest., 95, 577-585. [7] Mudde, G.C., Bheekha, R. & Bruijnzeel-Koomen, C.A.F.M. (1995), IgE-mediated antigen presentation. Aller3!y, 50, 193-199. [8] Mudde, G.C., Bheekha, R. & Bmijnzeel-Koomen, C.A. (1995), Consequences of IgE/CD23-mediated antigen presentation in allergy. Immunol. Today, 16, 380-383. [9] Mudde, G.C., Hansel, T.T., V. Reijsen, F.C., Osterhoff, B.F. & Bruijnzeel-Koomen, C.A.F.M. (1990), IgE: An immunoglobulin specialized in antigen capture. lmrnunol. Today, 11,440-443. [10] Mudde, G.C., Reischul, I.G., Corvaia, N., Hren, A. & PoeUabauer, E.M. (1996), Antigen presentation in allergic sensitization, lmmunol. Cell BioL, 74, 167-173. [11] Santamaria, L.F., Bheekha, R., Van Reijsen, F.C., Perez Soler, M.T., Sut~;r, M., Bruijnzeel-Koomen, C.A.F.M. & Mudde, G.C. (1993), Antigen focusing by specific monomeric immunoglobulin E bound to CD23 on Epstein-Barr virus-transformed B cells. Hum. lmrnunol., 37, 23-30. [12] Van der Heijden, F.L., Van Neerven, R.J. & Kapsenberg, M.L. (1995), Relationship between facilitated allergen presentation and the presence of allergen-specific IgE in serum of atopic patients. Clin. Exp. L'nmunol., 99, 289-293. [13] Van der Heijden, F.L., Van Neerven, R.J.J., Van Katwijk, M., Bos, J.D. & Kapsenberg, M.L. (1993), Serum-IgE-facilitated allergen presentation in atopic disease. J. Irnmunol., 150, 3643-3650. [14] Van d,~r Zee, J.S. & Aalberse, R.C. (1987), The role of IgG in allergy, in: "Allergy : an international textbook". (Lessof, M.H., Lee, T.H., Kemeny, D.M.) (pp. 49-67) Wiley, London. [15] Van der Zee, J.S. & Aalberse, R.C. (1991), The role of IgG in immediate-type hypersensitivity. Eur. Respir J., 13, 91s-96s. [16] Van der Zee, J.S., Van Swieten, P. & Aalberse, R.C. (1986), Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency. J. lmmunol., 137, 3566-3571.