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. 2001 Oct;108(8):1151-8.
doi: 10.1172/JCI11494.

Mast Cells Play a Key Role in Neutrophil Recruitment in Experimental Bullous Pemphigoid

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Free PMC article

Mast Cells Play a Key Role in Neutrophil Recruitment in Experimental Bullous Pemphigoid

R Chen et al. J Clin Invest. .
Free PMC article

Abstract

Bullous pemphigoid (BP) is an inflammatory subepidermal blistering disease associated with an IgG autoimmune response to the hemidesmosomal protein BP180. Passive transfer of antibodies to the murine BP180 (mBP180) ectodomain triggers a blistering skin disease in mice that depends on complement activation and neutrophil infiltration and closely mimics human BP. In the present study, we show that mast cells (MCs) play a crucial role in experimental BP. Wild-type mice injected intradermally with pathogenic anti-mBP180 IgG exhibited extensive MC degranulation in skin, which preceded neutrophil infiltration and subsequent subepidermal blistering. In contrast, mice genetically deficient in MCs or MC-sufficient mice pretreated with an inhibitor of MC degranulation failed to develop BP. Further, MC-deficient mice reconstituted in skin with MCs became susceptible to experimental BP. Despite the activation of complement to yield C3a and C5a, in the absence of MCs, accumulation of neutrophils at the injection site was blunted. The lack of response due to MC deficiency was overcome by intradermal administration of a neutrophil chemoattractant, IL-8, or by reconstitution of the injection sites with neutrophils. These findings provide the first direct evidence to our knowledge that MCs play an essential role in neutrophil recruitment during subepidermal blister formation in experimental BP.

Figures

Figure 1
Figure 1
Time-course of MC degranulation in MC-sufficient mice injected intradermally with pathogenic rabbit anti-murine BP180 IgG and assessed 0–12 hours after injection. (a) Toluidine blue–stained sections showing progression of MC degranulation and separation of the epidermis from the dermis. d, dermis; e, epidermis; v, vesicle; arrow, site of antibody labeling. ×400. (b and c) Electron micrographs show degranulation of MCs in dermis of mice injected with pathogenic IgG. An MC in control animals (b) has a number of large cytoplasmic granules that appear in different density, whereas another MC from pathogenic IgG-injected animal (c) presents many empty vacuoles that apparently resulted from degranulation. Numerous microvilli are observed in the plasma membrane of degranulated MCs, indicating the activated cell status. (d) The MCs in the dermis were counted and classified as degranulated (>10% of the granules exhibiting fusion or discharge) or normal (see Methods). MC degranulation reached the peak level at 1–2 hours after pathogenic IgG injection. (e) Skin MPO activity (mean ± SEM) data reveal the onset of PMN infiltration at 2 hours after IgG injection (n = 5 for each group shown).
Figure 2
Figure 2
Clinical and immunohistological analysis of neonatal MC-deficient and -sufficient mice injected with pathogenic anti-mBP180 IgG. The anti-mBP180 IgG (2.5 mg/g body weight) induced extensive blistering disease in MC-sufficient (+/+) mice (a). The skin of these animals showed linear deposition of rabbit IgG (b) and murine C3 (c) at the BMZ, as determined by direct IF. Toluidine blue staining revealed epidermal-dermal separation with MC degranulation (d). In contrast, MC-deficient (MgfSl/MgfSl-d) mice injected intradermally with pathogenic IgG showed no evidence of skin disease (e). Direct IF demonstrated BMZ deposition of rabbit IgG (f) and murine C3 (g). Toluidine blue staining showed no epidermal-dermal separation and absence of MCs (h). d, dermis; e, epidermis; v, vesicle; black arrow in a and e, site of clinical blister; white arrow, site of antibody labeling; black arrow in d and h, site of BMZ. ×400. Inset in d is a lower-magnification micrograph showing the edge of a subepidermal vesicle. ×100.
Figure 3
Figure 3
Effect of MC reconstitution on neutrophil infiltration and subsequent subepidermal blistering in pathogenic anti-BP180 IgG-injected MC-deficient mice. Neutrophil infiltration and blister formation were determined in the left (MC-deficient) (middle bar) and right (MC-reconstituted) (bottom bar) ears of Kitw/Kitw-v mice that underwent local reconstitution of the right ear with 1 × 106 bone marrow-derived MC+/+ MCs10 weeks before pathogenic IgG injection (2 mg/ear). MC+/+ littermates (top bar) were also injected with the same dose of IgG in the ear as positive control (see Methods for details). Significantly elevated levels of MPO activity (mean ± SEM) were seen in the right ear compared with the left ear of Kitw/Kitw-v mice. Like MC+/+ mice, the right but not the left ear skin sections exhibited dermal-epidermal separation as determined by H&E staining. MPO activity at 0 hours was 0.07 ± 0.01 for MC-deficient and 0.09 ± 0.02 (OD460nm/mg protein) for MC+/+ mice. (n = 6 for each group.) *P < 0.01, top vs. middle bar.
Figure 4
Figure 4
Experimental BP in MC-deficient mice reconstituted with normal PMNs. Pathogenic rabbit anti-mBP180 IgG (intradermal injection, 2.5 mg/g body weight) induced skin blisters in MC-sufficient (+/+) mice (a) and toluidine blue staining showed the presence of MCs (b). Anti-mBP180 IgG failed to trigger the skin lesions in MC-deficient MgfSl/MgfSl-d mice (c), but R621 IgG triggered the skin disease in MgfSl/MgfSl-d mice reconstituted with PMNs from MC+/+ mice (e) or MC-deficient mice (data not shown). Toluidine blue staining confirmed the MC deficiency in these animals (d and f).
Figure 5
Figure 5
In vivo reconstitution of PMNs at the tissue site by intradermal injection of IL-8 restores the pathogenic effect of anti-BP180 IgG in neonatal MC-deficient mice. MC-sufficient (+/+) (bar 1) or MC-deficient MgfSl/MgfSl-d mice (bars 2–6) were injected intradermally with either pathogenic anti-mBP 180 IgG alone (bars 1 and 4), control rabbit IgG alone (bar 2), hIL-8 alone (bar 3), or IgG plus hIL-8 (bars 5 and 6). MC-deficient mice coinjected with IL-8 and anti-mBP 180 IgG (bar 5), but not the control rabbit IgG (bar 6), developed subepidermal blisters. The IgG dose was 2.5 mg/g body weight. Doses for IL-8 was 50 ng/mouse. Tissue MPO activity (mean ± SEM) in skin at the injection site was determined 12 hours after IgG administration. Each group of mice (n = 5) without injection yielded an average of MPO activity of 0.08 ± 0.02 OD460nm/mg protein. *P < 0.01, Student’s t -test for paired samples (bar 4 versus bar 5). See Methods for details.
Figure 6
Figure 6
MC degranulation depends on complement activation. Neonatal C5-sufficient (a and b) and C5-deficient mice (c and d) were injected intradermally with pathogenic IgG R621 (2.5 mg/g body weight). C5+ (b) but not C5 mice (d) developed subepidermal blisters 12 hours after IgG injection. Toluidine blue staining analysis showed MC degranulation in the skin of C5+ mice at 2 hours (a) and 12 hours (b), whereas minimal levels of MC degranulation were seen in the dermis of C5 mice (b and d). d, dermis; e, epidermis; v, vesicle; arrow, site of basal keratinocyte. Original magnification ×400 (a and b), ×200 (c and d).
Figure 7
Figure 7
MPO activity of skin extracts from mice injected intradermally with pathogenic rabbit anti-mBP180 IgG. Neonatal MC-deficient MgfSl/MgfSl-d (bars 1 and 2), Kitw/Kitw-v (bars 3 and 4), and congenic normal (+/+) mice (bars 5 and 6) received 2.5 mg/g body weight pathogenic anti-mBP180 IgG. Tissue MPO activities (mean ± SEM) in the injection sites were determined 4 hours (bars 1, 3, and 5) and 12 hours (bars 2, 4, and 6) after the IgG injection (n = 5 for each group). *P < 0.01; **P < 0.005. Student’s t test for paired samples (bar 2 or 4 versus bar 6). The MPO values shown were corrected for control IgG controls. Each group of mice injected with control IgG yielded an average MPO activity of ∼0.13 OD460nm/mg protein.

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