FUNDAMENTAL AND APPLIED TOXICOLOGY 19,479-483 (1992)
SYMPOSIUM OVERVIEW
Chemical Allergy: Molecular Mechanisms
IAN KIMBER,* G. FRANK GERBERICK,~ HENK VAN LOVEREN,*
*ICI Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ. United Kingdom; iHuman and Environmental Safety Division,
The Procter and Gamble Company, Miami Valley Laboratories, PO Box 398107. Cincinnati, Ohio, 45239-8707; SLaboratory for Pathology,
National Institute of Public Health and Environmental Protection, 3720 BA Bilthoven, The Netherlands: and
$Life Sciences Research, IIT Research Institute, 10 West 35th Street, Chicago, Illinois 60616
Received
June IO.
ChemicalAllergy: Molecular Mechanismsand Practical Ap-
plications. KIMBER, I., GERBERICK, G. F., VAN LOVEREN, H.,
AND HOUSE, R. V. (1992). Fundam. Appl. Toxicol. 19,419-483.
Allergic reactions can be defined as the adverse, tissue-dam-
aging, and sometimesfatal consequencesof specific immune
responses,usually to exogenousantigens.In the context of tox-
icology it is allergic reactions resulting from immune responses
to chemicals and drugs which are of greatest relevance. The al-
lergy may take a variety of forms including contact hypersen-
sitivity (allergic contact dermatitis), respiratory hypersensitivity
(with symptomsranging from mild rhinitis to severeasthma),
and various types of comparatively ill-defined reactions which
in many respectsresembleautoimmunity. Of thesecontact hy-
persensitivityis the most frequently encounteredhealth problem
resulting from the interaction of chemicalswith the immune
system.A wide variety of chemicals are able to induce contact
sensitization. Some of these are, in addition, known to cause
respiratory hypersensitivity, a lessfrequent, but no lessimpor-
tant, form of chemicalallergy. o 1992Society of Toxicology.
A symposium entitled Chemical Allergy: Molecular
Mechanisms and Practical Applications was held at the 3 1st
Annual Meeting of the Society of Toxicology (SOT) in Seat-
tle, Washington in 1992. This symposium, sponsored by the
Immunotoxicology Specialty Section of the SOT, sought to
review selected aspects of the immunobiological mechanisms
which result in the development and expression of contact
and respiratory hypersensitivity and to identify, where pos-
sible, applications of value to investigative toxicology.
The Induction and Regulation of Immune Responses to
Chemical Allergens (Ian Kimber)
An intriguing question is why chemicals vary with respect
to the type(s) of allergic reactions they will preferentially elicit.
’ Symposium held at the 3 1stAnnual Meeting of the Society of Toxicology
(SOT), Seattle, WA. Sponsored by the Immunotoxicology Specialty Section
of the SOT.
and Practical Applications’
AND ROBERT V. HOUSES
1992;accepted
June 11, 1992
Dearman and Kimber (199 1) and Dearman et al. ( 199 1,
1992b) have examined the characteristics of immune re-
sponses induced in mice by trimellitic anhydride (TMA) and
2,4-dinitrochlorobenzene (DNCB). TMA is a known human
respiratory allergen which also has the potential to cause
contact hypersensitivity. In contrast, DNCB is a potent con-
tact allergen which fails to induce respiratory hypersensitivity.
Of particular interest is the relative ability of TMA and
DNCB to stimulate the production of IgE, the class of an-
tibody known to cause immediate hypersensitivity reactions
in the respiratory tract. Under conditions of topical exposure
where both chemicals were immunogenic, each causing
equivalent levels of T lymphocyte proliferation in lymph
nodes draining the site of exposure, contact hypersensitivity
and comparable total IgG anti-hapten antibody responses,
only TMA provoked an increase in the serum concentration
of IgE and the appearance of specific IgE antibody (Dearman
and Kimber, 1991).
It was considered that the divergent immune responses
induced by these chemicals might reflect the differential ac-
tivation of distinct populations of T helper (TH) cells. A
functional heterogeneity among TH cells in mice has been
described by Mosmann et al. (1986) who recognized two
main populations, designated TH1 and THZ, which differ with
respect to the spectrum of cytokines they secrete following
activation. Although both populations produce interleukin
3 (IL-3) and granulocyte/macrophage colony-stimulating
factor (GM-CSF), only TH1 cells secrete interleukin 2 (IL-
Z), tumor necrosis factor p (TNF-& lymphotoxin), and in-
terferon y (IFN-7) and only THZ cells secrete interleukins 4,
5, 6, and 10 (IL-4, IL-5, IL-6, and IL-IO) (Mosmann and
Coffman, 1989; Mosmann et al., 199 1). More recently a
similar, but possibly not identical, functional heterogeneity
among human TH cells has been described (Parronchi et al.,
199 1; Romagnani, 199 1). Although the situation is complex
inasmuch as THI and TH2 cells may represent the most dif-
ferentiated forms of TH lymphocytes and derive from com-
mon precursors (Mosmann et al., 1991), this functional di-
479 0272-0590192$5.00
Copyright 0 1992 by the Society of Toxicology.
All rights of reproduction in any form reserved.
480 KIMBER
chotomy has considerable relevance for the development of
allergy, and in particular for the development of respiratory
hypersensitivity. The initiation and maintenance of IgE re-
sponses is dependent upon the availability of IL-4, a product
of Tu2 cells (Finkelman et al., 1988b). In contrast, IFN-7
produced by TnI cells is known to inhibit IgE antibody re-
sponses (Finkelman et al., 1988a). The information available
indicates, therefore, that Tn, and THZ cells and their soluble
products exert reciprocal antagonistic effects on IgE antibody
and possibly also on delayed-type hypersensitivity, including
contact hypersensitivity, responses.
Evidence for the differential stimulation of Tn subpopu-
lations by TMA and DNCB derives from analysis of the
isotype distribution of IgG antibody provoked by exposure
to these chemicals. In addition to inhibiting IgE antibody
production, IFN-7 (but not Tm cell cytokines) has been
shown to augment IgG,, responses (Finkelman et al., 1988a).
Consistent with a preferential activation of Tn, cells, DNCB
was found to stimulate high levels of IgG,, antibody (Dear-
man and Kimber, 199 1). Conversely, and consistent with a
preferential activation of Tm-type responses, TMA provoked
only low levels of IgG2, (Dearman and Kimber, 1991). Sub-
sequent studies have revealed that other known human re-
spiratory allergens such as, for instance, diphenylmethane-
4,4’-diisocyanate and phthalic anhydride also induce in mice
immune responses characteristic of Tu2 cell activation
(Dearman and Kimber, 1992; Dearman et al., 1992~). Other
chemicals, including 4-ethoxymethylene-2-phenyloxazol-5-
one (oxazolone), dicyclohexylmethane-4,4’-diisocyanate, and
isophorone diisocyanate, which are known or suspected not
to induce respiratory hypersensitivity, failed to stimulate IgE
antibody and provoked immune responses consistent with
a selective, and in some casespossibly an exclusive, activation
of Tu,-type responses (Dearman and Kimber, 1992; Dear-
man et al., 1992~).
Clearly a preferential, rather than exclusive, activation of
THZ cells by respiratory allergens accommodates the fact that
the majority, if not all, chemicals which are able to cause
respiratory hypersensitivity appear also to be capable of in-
ducing contact sensitization, albeit weakly in some cases.
The reasons why chemicals appear to differ with respect
to the selectivity of Tu cell responses remain unresolved.
Nevertheless, the characteristics of allergen-induced immune
responses, and in particular the differences in immune re-
sponses induced by contact and respiratory allergens, is po-
tentially of considerable utility in predictive toxicology. As
yet there exists no well-validated or widely applied method
for the prospective identification of chemical respiratory al-
lergens. To date, attempts to develop predictive methods
have focused on the guinea pig and the measurement of
inhalation-induced respiratory responses in previously sen-
sitized animals. (Karol et al., 1981; Botham et al., 1989;
Pauluhn and Eben, 199 1; Sarlo and Clark, 1992.) An alter-
native approach, the mouse IgE test, is based upon the ob-
ET AL.
servation that, of the chemicals examined to date, only those
with a known potential to induce respiratory sensitization
caused an increase in the serum concentration of IgE; a con-
sequence presumably of IL-4 production. Exposure of mice
to immunogenic concentrations of contact allergens which
apparently lack the ability to cause respiratory hypersensi-
tivity failed to influence the concentration of IgE in serum
(Dearman et al., 1992a). Currently this test method is being
validated with a wider range of chemicals.
The Processing and Presentation of Chemical Allergens
(G. Frank Gerberick)
The skin is an important site for the development of
chemical allergy being, by definition, the route of exposure
for contact sensitization. It is likely also that dermal exposure
to chemical allergens can induce immune responses resulting
in sensitization for respiratory allergy.
It is now apparent that epidermal Langerhans cells (LC)
play a critical role in skin sensitization. The available evi-
dence indicates that following topical exposure to sensitizing
chemicals the allergen associates with LC (Shelley and Juhlin,
1976) and that these cells are induced to migrate, and trans-
port antigen, via the afferent lymphatics, to the draining
lymph nodes (Silberberg-Sinakin et al., 1976; Macatonia et
al., 1986; Cumberbatch and Kimber, 1990). Although it is
known that LC are able to process protein antigen for sub-
sequent presentation to responsive T lymphocytes (Streilein
et al., 1990) their activity in this respect with chemical al-
lergens is not known. The recognition and processing by LC
of chemical sensitizers have been studied indirectly using the
photoallergen, tetrachlorosalicylanilide (TCSA) (Gerberick
et al., 199 1b). Following ultraviolet (UV) irradiation TCSA
can associate with protein; a process that may be examined
visually by fluorescence (Kochevar and Harber, 1977). The
binding of TCSA to epideimal cells (EC) following UVA
irradiation in vitro has been measured using fluorescence
microscopy and image analysis. The irradiated chemical was
shown to associate with both LC and other epidermal cells,
presumably keratinocytes (Gerberick et al., 199 1b). The peak
fluorescence intensity of LC-enriched EC treated with TCSA
and UVA occurred following excitation at a wavelength of
380 nm. In contrast, the same populations treated with unir-
radiated TCSA or irradiated with UVA prior to TCSA treat-
ment were found to display maximum fluorescence when
excited at 340 nm (Gerberick et al., 199 1b). It is not yet clear
whether the altered characteristics of TCSA following inter-
action with LC reflect active processing of the chemical al-
lergen by LC.
Irrespective of the need for allergen processing by LC,
it is clear that the mobilization of these cells following
contact sensitization and their migration from the skin,
results in an influx of antigen-bearing dendritic cells (DC)
into lymph nodes draining the site of exposure (Silberberg-
Sinakin et al.. 1976; Macatonia et al., 1986; Cumberbatch
 CHEMICAL ALLERGY: MECHANISMS
and Kimber, 1990; Kripke et al., 1990). A similar increase
in the frequency of lymph node DC has been found fol-
lowing contact photosensitization with TCSA (Gerberick
et al., 1991a). The antigen borne by the DC which accu-
mulate in draining lymph nodes is highly immunogenic.
Antigen-bearing DC will efficiently transfer contact sen-
sitization to naive recipients and will stimulate prolifer-
ative responses by lymphocytes in vitro. To investigate the
presentation of chemical allergen to T lymphocytes the
latter phenomenon has been measured using a blastoge-
nesis assay. The ability of antigen-bearing DC isolated
from the draining lymph nodes of sensitized mice, and of
LC-enriched populations of EC modified with hapten in
vitro, to stimulate proliferative responses by lymphocytes
prepared from previously sensitized mice was examined.
Draining lymph node DC isolated from mice 24 hr fol-
lowing treatment with the allergen picryl chloride were found
to induce strong proliferative responses by lymphocytes de-
rived from mice sensitized previously with the same chem-
ical. The response was shown to be antigen-specific insofar
as DC prepared from mice treated with an unrelated allergen,
or prepared from naive mice, were significantly less stimu-
latory (Gerberick et al., 1992). Similarly, specific proliferative
responses were observed following culture of lymphocytes
with DC isolated from the lymph nodes of mice treated 24
hr previously with TCSA and UVA (Gerberick et al., 199 1a).
DC prepared from mice treated with UVA prior to TCSA
were nonstimulatory. Hapten (picryl chloride)-modified LC-
enriched EC populations were found also to induce specific
proliferative responses by lymphocytes prepared from mice
treated with the same chemical (Robinson et al., 1989).
Moreover, LC prohapten-modified by TCSA and UVA were
found capable of inducing proliferation by lymphocytes
drawn from TCSA-photosensitized mice. Untreated LC, and
LC treated with UVA prior to TCSA, were without effect
(Gerberick et al., 1991b).
Such blastogenesis assays which measure secondary pro-
liferative responses are of value in confirming and investi-
gating sensitization and photosensitization and in examining
cross-reactivity between allergens. Of considerable interest
is the fact that, in addition to inducing secondary proliferative
responses, epidermal LC and lymph node DC are sufficiently
stimulator-y to provoke primary proliferative responses in
vitro by lymphocytes derived from naive mice (Macatonia
et al., 1986; Hauser and Katz, 1988). Such may provide one
way forward for the development of in vitro methods to ex-
amine the sensitization potential of chemicals.
A Role for Cellular Immunity in the Induction of Airway
Hyperreactivity by Small Mo/ecuiar Weight Compounds
(Henk Van Loveren)
Clinically, the symptoms associated with respiratory
hypersensitivity responses to chemicals are various and
may include both immediate-onset and late-phase reac-
AND APPLICATIONS 481
tions. Immediate responses, including acute-onset asth-
matic reactions, are regarded generally as being attribut-
able to IgE antibody-mediated degranulation of tissue mast
cells and the release of vasoactive amines and mediators
such as leukotriene B4 (LTB4) which result in broncho-
constriction, In some cases, however, airway hyperreac-
tivity persists or displays a late-onset occurring 2 to 8 hr
following exposure. Such late-phase reactions are not in-
frequently associated with an infiltration of mononuclear
cells and there is evidence that chronic T cell-mediated
immune responses in bronchial tissue may mediate per-
sistent airway hyperreactivity (Poulter et al., 1990). Recent
attempts to examine the role of T lymphocytes in allergic
airway hyperresponsiveness have made use of a murine
model for measuring cellular immunity in pulmonary tis-
sue (Enander et al., 1983, 1988). Mice exposed previously
by topical application of picryl chloride were challenged
by intranasal exposure to picryl sulfonic acid, an antigen-
ically cross-reactive, water-soluble derivative of the aller-
gen. Such challenge resulted in an accumulation of mono-
nuclear cells around bronchioli and blood vessels. This
response was maximal after 48 hr and characteristic of a
delayed-type hypersensitivity (DTH) reaction. The in-
flammatory responses were found to be dependent upon
serotonin and a cascade of cellular events such as those
shown previously to occur during DTH reactions in the
skin: an increase in vasopermeability followed by mono-
nuclear cell infiltration (Garssen et al., 1989). This pul-
monary DTH reaction was shown to be associated with
alterations in lung function. However, airway hyperreac-
tivity, as measured by the responsiveness of murine trachea
to the smooth muscle contractant carbachol, preceded and
persisted much longer than the cellular infiltrate (Garssen
et al., 199 1; Van Loveren et al., 1991). The rapid devel-
opment of tracheal hyperresponsiveness is considered to
be due to a reaction resulting from the release of vasoactive
amines and mediated by a subset of T lymphocytes shown
previously to cause early (l-2 hr) oedematous reactions
in cutaneous hypersensitivity responses (Van Loveren et
al., 1983; Van Loveren and Askenase, 1984; Herzog et al.,
1989). The importance of mast cells in this process is sug-
gested by the observation that nedocromil, a drug which
inhibits mediator release from mast cells, suppressed the
T cell-dependent tracheal hyperresponsiveness to car-
bachol.
It is concluded that both major subpopulations of T helper
cells have the potential to influence the development of al-
lergic pulmonary reactions. Induction of Tuz-type responses
and IL-4 release will facilitate IgE antibody production and
sensitization for acute pulmonary reactions. In addition Tu,
cells, which are known to effect DTH reactions (Cher and
Mosmann, 1987), may also participate in pulmonary hy-
persensitivity reactions and be of particular significance in
late-onset responses.
482 KIMBER ET AL.

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