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*
Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University of Münster, Münster, Germany;
Departments of Surgery and Physiology, University of California San Francisco, San Francisco, CA 34143;
Department of Dermatology, Emory University School of Medicine, Atlanta, GA 30322;
Institute of Pharmacology, University of Ferrara, Ferrara, Italy;
¶ Ina Sue Perlmutter Laboratory, Children’s Hospital, Departments of Pediatrics and Medicine, Harvard Medical School, Boston, MA 02115; and
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Department of Dermatology, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biological actions of SP are terminated by neutral endopeptidase (NEP). NEP is a member of a family of cell surface zinc metalloproteinases that also includes endothelin-converting enzyme and the Kell blood group Ag (8, 9). NEP is expressed in several tissues including the kidney, small intestine, brain, airway epithelium, vascular endothelium, and skin (8). The administration of NEP inhibitors augments inflammation of the trachea (10, 11). NEP expression is markedly down-regulated during intestinal and respiratory infections, which may contribute to the tissue inflammation response in these diseases (12, 13). Deletion of NEP expression by targeted disruption of the NEP gene locus in mice results in an increased susceptibility to endotoxin shock and intestinal inflammation as well as to the induction of hypotension and a widespread plasma extravasation from postcapillary venules that is mediated by the NK1R and the bradykinin 2 receptor (14, 15).
Little is known about the relative contributions of the neurological system, SP, NK1R, and NEP in inflammatory skin disease. In this study, the role of NEP in modulating cutaneous inflammation in expression of allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) responses was examined. We tested the hypothesis that the absence or inhibition of NEP results in a dysregulated inflammatory skin response in these skin diseases. To address this possibility, we measured the relative contribution of cutaneous NEP in an experimental model of ACD compared with ICD using NEP-/- mice and corresponding wild-type animals. Our results indicate that the lack of NEP results in an increased cutaneous inflammatory response to challenge by allergens, but not by irritants. These results implicate the SP proteolytic enzyme NEP as an important modulator of ACD inflammatory responses in the skin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NEP-/- mice were a gift from Dr. Craig Gerard (Harvard University, Boston, MA) (14). These mice were derived from the C57BL/6 strain with seven backcrosses. NEP+/+ C57/BL6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and used as the control strain. Mice were housed in a barrier facility with free access to water and food. Males and females were studied at 8–12 wk with five to seven mice per experimental condition.
Experimentally induced ACD and administration of neuromodulating agents
ACD was induced as previously described (16). Mice were sensitized on day 0 by painting 25 µl of 0.5% 2,4-dinitro-1-fluorobenzene (DNFB; Sigma, St. Louis, MO) in acetone/olive oil (4:1) on the shaved abdomen. Mice were challenged on day 5 by epicutaneous application of 10 µl 0.2% DNFB in acetone/olive oil (4:1) on the dorsal surface of the right ear. The left ears were treated with vehicle alone (acetone/olive oil, 4:1) and served as an internal control for these studies. The ear swelling induced by the carrier alone was negligible. To detect any potential irritant effects of the hapten, some mice were challenged with 10 µl 0.2% DNFB on the right ear without prior sensitization. The ACD response was determined by the degree of ear swelling of the hapten-exposed ear compared with that of the vehicle-treated contralateral ear before DNFB challenge and at 0–72 h after challenge, as measured with a micrometer.
Ears were obtained from euthanized mice and processed for histologic examination. In some experiments, the effect of neuropeptide depletion on ACD was determined by application of the sensory neurotoxin capsaicin (Zostrix HP cream; GenDerm, Lincolnshire, IL) containing 0.075% trans-8-methyl-N-vanillyl-6-nonenamide (capsaicin) to the right ears of sensitized NEP-/- mice twice a day for 3 days before Ag challenge and then at multiple times after challenge (15 min, and 7, 14, and 24 h). As a control, the left ears of these mice were treated at each time point with vehicle alone. In a separate study, mice were treated with Zostrix HP cream vehicle only, which was prepared according to the formula provided by GenDerm. In the capsaicin studies, the ear thickness of both the right and left ear of each mouse was measured at 24, 48, and 72 h after challenge. To determine the effect of acute NEP inhibition on ACD in wild-type animals, the NEP inhibitor phosphoramidon (Sigma) was injected into the tail veins of anesthetized sensitized NEP+/+ mice at a dose of 2.5 mg/kg at 1, 4, and 8 h after Ag challenge. The effect of SP receptor blockage on ACD was measured by administration of the NK1R antagonist SR 140333 (1.2 µmol/kg, i.v.) (Sanofi Recherche; Montpellier, France) to anesthetized sensitized NEP-/- mice at 2 and 6 h after Ag challenge. All studies were conducted two to three times to verify reproducibility of results.
Induction of ICD
After measurement of the ear thickness of untreated animals, mice were treated with 10 µl of the cutaneous irritants croton oil in acetone (0.25, 0.8, or 1.6%; Sigma) or SDS in distilled water (2 or 10%) on the dorsal surface of the right ear to induce ICD as previously described (17). The left ear was treated with vehicle only. The ICD response was determined by the degree of ear swelling of the irritant-exposed ear compared with that of the vehicle-treated contralateral ear before irritant challenge and at 0–48 h after challenge as measured with a micrometer. From some mice of each group ears were harvested after 24 h and processed for histologic evaluation. Experiments were performed twice to verify reproducibility of the results.
Measurement of microvascular permeability
ACD was induced as previously described (16) in NEP-/- and NEP+/+ mice. Microvascular permeability was determined by cutaneous Evans blue extravasation. To measure extravasation of Evans blue, anesthetized mice were injected with 30 mg/kg Evans blue (Sigma) in 0.9% NaCl into a femoral vein (18). Seven minutes after Evans blue injection, mice were transcardially perfused with 50 ml PBS containing 100 U/ml heparin and 200 ml 1% paraformaldehyde in a 0.05% citrate buffer, pH 3.5. Ears were removed, rinsed in saline, blotted, and weighed. Half of each tissue was dried at 60°C for 48 h and reweighed; the other half was incubated in 1 ml formamide (Sigma) for 48 h at room temperature to extract the Evans blue. Evans blue was quantified by spectrophotometry (18). Tissue extravasation is expressed as nanograms Evans blue per milligram of dry weight of tissue. This study was conducted twice with similar results.
Histology
Ears were fixed in 10% PBS-buffered formalin, and 3- to 5-µm paraffin sections were stained with hematoxylin and eosin and examined by light microscopy to assess histologic changes and immune cell infiltration.
Immunofluorescence
Ears were fixed in 4% paraformaldehyde for 24 h at room temperature and embedded in paraffin or snap frozen, embedded in OCT compound (Miles, Elkhart, IN), sectioned, and fixed in 4% paraformaldehyde for 20 min. Sections were processed for immunofluorescence (19). Slides were incubated with a rabbit antiserum to recombinant human NEP (1:250, overnight, 4°C) (20). Endothelial cells were identified using an Ab to von Willebrand factor (1:100; Dako, Carpinteria, CA).
Statistical analysis
Results are expressed as mean ± SE. Differences between multiple groups were examined using an ANOVA and Bonferroni t test. Mean differences with p < 0.05 were considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunofluorescence studies were conducted to determine the
distribution of NEP in normal skin and confirm that NEP expression was
absent from NEP-/- mice. In
NEP+/+ animals, NEP was immunolocalized in the
keratinocytes of the epidermis (Fig. 1
B), in dermal microvascular
endothelial cells (Fig. 1D), and in hair follicles (data not
shown). As expected, NEP was not detected in the skin of
NEP-/- mice (Fig. 1, A and
C).
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To assess the role of NEP in modulating cutaneous inflammation,
we compared the ACD response in NEP-/- to
NEP+/+ mice. In DNFB-sensitized mice, application
of DNFB to the right ear induced marked swelling in both the
NEP+/+ and NEP-/- animals
(Fig. 2). Notably, the response was
significantly higher (80–120%) in NEP-/-
compared with NEP+/+ mice at all time points.
DNFB challenge in nonsensitized animals failed to induce ear swelling
in either the NEP-/- or
NEP+/+ mice, indicating that the reaction to DNFB
was allergic and not due to an irritant effect of the DNFB vehicle
solution.
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We have previously demonstrated that
NEP-/- mice have increased permeability of
postcapillary venules under basal conditions (18). To
determine whether this lack of NEP causes increased microvascular
permeability during the expression of ACD, we challenged sensitized and
nonsensitized NEP-/- and
NEP+/+ mice with DNFB and measured extravasation
of plasma proteins labeled with Evans blue. Without prior
sensitization, DNFB has little effect on extravasation of Evans blue in
NEP-/- and NEP+/+ mice
(Fig. 3, a and b).
In sensitized animals, DNFB induced marked plasma extravasation in
NEP-/- and NEP+/+
compared with carrier-treated control ears (Fig. 3, c and
d). Extravasation at 6 h after Ag challenge was
significantly higher in NEP-/- mice compared
with the NEP+/+ animals, indicating that this
critical component of the ACD response acts early in this cutaneous
inflammatory response (Fig. 3, c and d). These
results indicate that cutaneous plasma extravasation regulated by NEP
is an important early component of the efferent phase of ACD.
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We tested the possibility that augmented efferent ACD responses
and extravasation of plasma proteins during ACD in
NEP-/- mice was due to diminished degradation
of cutaneous SP. First we tested the potential of the neurotoxin
capsaicin to inhibit the augmented ACD response in
NEP-/- animals by depleting the skin of
neuropeptides in sensory nerves. Zostrix HP cream (capsaicin) was
applied to the skin multiple times before and after Ag challenge.
Pretreatment of the skin with capsaicin before DNFB challenge slightly
increased the swelling, which was likely due to the initial
proinflammatory effects of capsaicin-induced neuropeptide release (data
not shown). After Ag challenge, we observed a profound reduction of the
cutaneous inflammatory response at 24 and 48 h in
NEP-/- mice treated with capsaicin compared
with NEP-/- animals not treated with capsaicin
(Fig. 4, a and b).
Animals treated with Zostrix vehicle alone demonstrated no inflammatory
response (data not shown). Therefore, the elevated ACD response
observed in NEP-/- mice can be inhibited by the
neurotoxin capsaicin, thus supporting the role of cutaneous
neuropeptides and an intact functional peripheral sensory nervous
system in ACD.
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, a and
b). These results indicate that the augmented ACD response
in NEP-/- mice is dependent on SP activation
of NK1R.
To determine whether acute inhibition of NEP in
NEP+/+ mice also increased the ACD response, as
in the NEP-/- animals, we treated
NEP+/+ mice with the selective NEP inhibitor
phosphoramidon. Intravenous administration of phosphoramidon at 1, 4,
and 8 h after DNFB challenge of NEP+/+ mice
caused a marked increase in the ACD response in a similar fashion as we
previously observed in the NEP-/- animals (Fig. 5). Phosphoramidon did not effect
cutaneous inflammation in nonsensitized animals (data not shown). Thus,
the acute inhibition of NEP in wild-type animals results in augmented
expression of ACD responses, which further supports the role of this
SP-degrading enzyme in Ag-mediated cutaneous inflammation.
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We further characterized the effector phase of ACD in
NEP-/- and NEP+/+ mice by
cutaneous histological examination. No significant histological
differences were observed in the skin of NEP-/-
and NEP+/+ animals treated with vehicle only
(Fig. 6, A and E).
Likewise, no differences in tissue histology were observed between
DNFB-challenged ears of nonsensitized NEP-/-
and NEP+/+ mice (data not shown). In sensitized
NEP-/- mice, we observed significant dermal
edema, a massive inflammatory infiltrate consisting predominantly of
neutrophils and mononuclear cells, and epidermal hyperplasia 16 and
72 h after Ag challenge (Fig. 6, B and C).
Notably, edema, cellular infiltrate, and epidermal hyperplasia were
clearly less prominent at these time points after Ag challenge in
sensitized NEP+/+ mice (Fig. 6, F and
G). Treatment of sensitized NEP-/-
mice with an NK1R antagonist after DNFB challenge resulted in a
dramatic reduction in cutaneous edema, leukocyte infiltration, and
hyperplasia (Fig. 6D) so that the histology of these ears
was comparable to that of the control ears (Fig. 6A),
whereas the systemic treatment of sensitized
NEP+/+ mice with the NEP inhibitor phosphoramidon
after Ag challenge (Fig. 6H) markedly augmented the
histologic parameters of the ACD inflammatory response compared with
untreated animals (Fig. 6E). These results indicate that
NEP, SP, and NK1R play a significant role in modulating the histologic
inflammatory changes at sites of Ag challenge during the effector phase
of ACD.
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When NEP-/- and
NEP+/+ mice were treated with various
concentrations of the topically applied irritant croton oil (0.25, 0.8,
and 1.6%), the ear swelling response was dose dependently increased in
NEP knockout and wild-type animals, reaching a peak swelling 6–8 h
after croton oil challenge (Fig. 7, a–c). However, in contrast to the results observed during
ACD, the ICD response of NEP-/- animals to
croton oil was not significantly different from that in wild-type
NEP+/+ animals (Fig. 7). Histologically, the
cellular inflammatory response in ICD was also not visibly different in
NEP-/- mice compared with
NEP+/+ animals (Fig. 8, C and D).
Likewise, the ICD response to challenge with 2 or 10% SDS was not
significantly different in NEP-/- and wild-type
mice when measured from 0 to 48 h as indicated for the croton oil
studies (data not shown). Thus, NEP is an important inflammatory
modulator of ACD responses, but not ICD cutaneous inflammation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The local tissue bioactivity of SP appears to be tightly controlled by its release from sensory nerves, the presence of NK1R on target cells, and local expression of SP-degrading enzymes such as NEP (34). NEP is a potent regulator of in vivo neuropeptide biological responses (11, 35). The role of NEP in modulating systemic inflammatory response is supported by a recent study demonstrating that NEP-/- animals are highly sensitive to bacterial endotoxins and endotoxic shock (14). In addition to hypotension, NEP-/- mice demonstrated an increased basal level of plasma extravasation in various organs as compared with NEP+/+ animals. This increased plasma leakage in NEP-/- mice could be abrogated by the injection of recombinant NEP, the NK1R antagonist SR140333, or a bradykinin B2 receptor antagonist (18).
In this study, we used experimental murine models for ACD and ICD to study the relative contribution of NEP in these common types of cutaneous inflammation. Our results provide substantive evidence for a major role for SP, the NK1R, and NEP in ACD. ACD elicitation inflammatory responses were markedly greater in animals lacking NEP or in wild-type mice treated with a NEP inhibitor, which supports the physiologic contribution of NEP in down-regulating the neuroinflammatory component of ACD. Enhanced expression of the augmented ACD elicitation response observed in NEP-/- mice was prevented by depletion of SP from cutaneous sensory nerves with capsaicin or by administration of an NK1R antagonist, which demonstrates that diminished degradation of SP released from cutaneous sensory nerves in NEP-/- animals contributes to the efferent phase of allergic inflammatory responses in the skin. In contrast, no differences were noted in the ICD inflammatory responses of NEP-/- mice compared with wild-type animals. Thus, our data demonstrate that the absence of NEP causes augmented and therefore dysregulated inflammatory effector responses to allergens, but not to irritants such as croton oil or SDS in this murine model. This is a novel and somewhat unexpected observation that is in contrast to a number of previous studies that demonstrated an involvement of sensory nerves and neuropeptides such as SP in both types of inflammation (1, 24, 26, 27, 29, 30, 31, 36).
The precise mechanisms responsible for these differences between acute
irritant inflammation and the hapten-specific ACD responses in mice
with nonfunctional NEP is not entirely clear. There is evidence that
the individual properties of the particular irritants croton oil or SDS
may account for some of the ICD inflammatory responses (17, 37). Another possible explanation for the observed differences
between ACD vs ICD responses is that in ACD responses, in contrast to
ICD, the released cutaneous neuropeptides may directly or indirectly
act on infiltrating T lymphocytes, inducing them to proliferate and
secrete additional inflammatory mediators that can initiate a cascade
of subsequent inflammatory events in the skin. SP is capable of
directly augmenting T cell-mediated immune responses (23, 24). SP stimulates the proliferation of T lymphocytes
(38), enhanced IL-2 production (39), and
expression of T lymphocyte activation Ags such as IL-2 receptor
-chain (CD25) and RT1B MHC class II molecule (40).
Released SP may also modulate ACD responses by acting on macrophages
and APCs (41, 42, 43). For example, SP is capable of inducing
IL-12 production in LPS-treated murine macrophages (44).
This is of particular importance because a number of studies have
implicated IL-12 as an important mediator of cellular immunity. IL-12
is a potent costimulator for the development of Th1 cells
(45). This is accomplished by IFN-
induction in Th1
cells (46, 47) and promotion of Ag-dependent proliferation
of activated T lymphocytes (48). SP is also capable of
directly inducing T cell IFN- that is known to suppress IL-4 and the
development of a Th2 cell phenotype as well as of enhancing
mitogen-induced IFN- in human PBMC (49, 50). IL-12 has
been demonstrated to act as an important mediator and adjuvant for ACD
induction (51). Neutralization of IL-12 by Ab injection
prevented the induction or expression of ACD, but not the ICD response
elicited by croton oil in mice, suggesting that IL-12 participates in
ACD rather than ICD responses (52). Thus, excessive
cutaneous SP in NEP-/- mice may favor a
distinct cytokine environment involving IL-12 and IFN- that promotes
a Th1-mediated cellular immune response typical of ACD. These important
immunologic activities could explain in part why excessive SP may
promote an expression of ACD but not an ICD response.
This study does not directly address the role of NEP on the induction of ACD, which will be the focus of ongoing studies in our laboratory. Nonetheless, it is noteworthy that preliminary studies in our laboratory revealed that acute inhibition of the angiotensin-converting enzyme (ACE), another peptidase that is capable of degrading SP and the SP-inducing bradykinin, not only enhances the ACD response to DNFB in mice when systemically applied before hapten challenge (elicitation), but also when applied before sensitization (53). Thus, the basis of the exaggerated ACD response observed in mice with inhibited or functionally deleted SP-degrading peptidases is active during the initial contact with the hapten (afferent phase), which strongly suggests the possibility that increased SP is capable of boosting both the sensitization and elicitation phase of ACD. In ACD induction, it appears to be conceivable that SP may directly enhance functions of epidermal Langerhans cells or dermal dendritic APCs stimulating Ag presentation and/or migration into the regional lymph nodes. In favor of this hypothesis is a recent report indicating that a SP agonist promoted ACD induction and prevented or reversed hapten-specific tolerance induced by low-dose UV B radiation in vivo (26).
In summary, our data indicate that the absence or reduced activity of NEP, the primary proteolytic peptidase for SP, leads to the development of augmented inflammatory effector phase responses to allergens in a murine ACD model system, which may be due to the prolonged tissue presence of released SP in the inflamed tissue. Our observations further support the hypothesis that the expression and regulation of NEP, SP, and NK1R play an important role in inflammatory skin disease such as ACD. Thus, neuroinflammation is a significant component of the effector phase of ACD. Modulation of the neurocutaneous system may lead to novel therapeutic agents for inflammatory disease of the skin.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John C. Ansel, Department of Dermatology, Emory University, 5313 Woodruff Memorial Building, Atlanta, GA 30322.
3 Abbreviations used in this paper: SP, substance P; NEP, neutral endopeptidase; ACD, allergic contact dermatitis; ICD, irritant contact dermatitis; NK1R, neurokinin receptor 1; DNFB, 2,4-dinitro-1-fluorobenzene.
Received for publication December 13, 1999. Accepted for publication October 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-melanocyte-stimulating hormone induces hapten-specific tolerance in mice. J. Immunol. 156:473.[Abstract]
release by human blood monocytes and macrophages. J. Neuroimmunol. 82:126.[Medline]
by neonatal human CD4 T cells. J. Immunol. 151:1938.[Abstract]
immunoregulatory circuit. Ann. NY Acad. Sci. 840:532.[Medline]
production by human peripheral blood mononuclear cells. Regul. Pept. 19:355.[Medline]
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