The Eotaxin-1/CCR3 Axis and Matrix Metalloproteinase-9 Are Critical in Anti-NC16A IgE-Induced Bullous Pemphigoid

Abstract

Bullous pemphigoid (BP) is the most common autoimmune bullous skin disease of humans and is characterized by eosinophilic inflammation and circulating and tissue-bound IgG and IgE autoantibodies directed against two hemidesmosomal proteins: BP180 and BP230. The noncollagenous 16A domain (NC16A) of BP180 has been found to contain major epitopes recognized by autoantibodies in BP. We recently established the pathogenicity of anti-NC16A IgE through passive transfer of patient-derived autoantibodies to double-humanized mice that express the human high-affinity IgE receptor, FcεRI, and human NC16A domain (FcεRI/NC16A). In this model, anti-NC16A IgEs recruit eosinophils to mediate tissue injury and clinical disease in FcεRI/NC16A mice. The objective of this study was to characterize the molecular and cellular events that underlie eosinophil recruitment and eosinophil-dependent tissue injury in anti-NC16A IgE-induced BP. We show that anti-NC16A IgEs significantly increase levels of key eosinophil chemoattractants, eotaxin-1 and eotaxin-2, as well as the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) in the lesional skin of FcεRI/NC16A mice. Importantly, neutralization of eotaxin-1, but not eotaxin-2, and blockade of the main eotaxin receptor, CCR3, drastically reduce anti-NC16A IgE-induced disease activity. We further show that anti-NC16A IgE/NC16A immune complexes induce the release of MMP-9 from eosinophils, and that MMP-9-deficient mice are resistant to anti-NC16A IgE-induced BP. Lastly, we find significantly increased levels of eotaxin-1, eotaxin-2, and MMP-9 in blister fluids of BP patients. Taken together, this study establishes the eotaxin-1/CCR3 axis and MMP-9 as key players in anti-NC16A IgE-induced BP and candidate therapeutic targets for future drug development and testing.

Figure 1:. Eotaxin-1 is critical in anti-NC16A IgE-induced BP in FcεRI/NC16A mice.

Adult FcεRI/NC16A mice were injected in the convex pinnae with either anti-NC16A IgE or normal human control IgE in the presence or absence of eotaxin-1 neutralizing antibodies, eotaxin-2 neutralizing antibodies, or isotype control antibodies. Mice were then evaluated for 0–48h post-injection. (A) The lesional skin of anti-NC16A IgE-injected mice exhibited significantly higher levels of eotaxin-1 and eotaxin-2 compared to control IgE-injected mice. At each timepoint, eotaxin-1/α-NC16A IgE was compared to eotaxin-1/control IgE, and eotaxin-2/α-NC16A IgE was compared to eotaxin-2/control IgE. (B, C) Neutralizing antibodies directed against eotaxin-1, but not eotaxin-2, significantly attenuated the severity of skin disease induced by anti-NC16A IgE in FcεRI/NC16A mice; bar 1 vs. bar 2. (D) Significantly reduced numbers of eosinophils infiltrated the skin of mice treated with neutralizing antibodies directed against eotaxin-1, but not eotaxin-2; bar 1 vs. bar 2. n=6, *p<0.05, **p<0.01, paired t-test.

Figure 2:. The eotaxin-1/CCR3 axis is critical in anti-NC16A IgE induced BP in FcεRI/NC16A mice.

Adult FcεRI/NC16A mice were injected in the convex pinnae with anti-NC16A IgE in the presence or absence of the selective CCR3 antagonist SB-328437 and were examined 48h post-injection. Blockade of CCR3 significantly reduced disease severity clinically (A, B) and histologically (C), and reduced the number of eosinophils that infiltrated the skin (D). α-NC16A IgE + inhibitor was compared to α-NC16A IgE. n=4 for bar 1, n=8 for bar 2, n=6 for bar 3, *p<0.01, paired t-test. Green, anti-MBP for eosinophils; red, anti-BP180; blue, DAPI.

Figure 3:. Anti-NC16A IgE induce the release of MMP-9 in vivo and in vitro.

(A) Adult FcεRI/NC16A mice were injected in the pinnae with either anti-NC16A IgE or normal human control IgE and examined 48h post-injection. Gelatin zymography examination revealed both pro- and active MMP-9 in the lesional skin of mice injected with anti-NC16A IgE, but not control IgE. (B) Purified eosinophils from FcεRI/NC16A mice were incubated with either anti-NC16A or control IgE, in the presence or absence of recombinant NC16A (rNC16A), for 1 hour. Pro-MMP-9 was only detected in the cell culture supernatants of eosinophils that were incubated with anti-NC16A IgE in the presence of rNC16A by gelatin zymography (lane 5).

Figure 4:. MMP-9 is critical in anti-NC16A IgE-induced BP in FcεRI/NC16A mice.

Adult FcεRI/NC16A (WT) and MMP-9-deficient FcεRI/NC16A (MMP-9 KO) mice were injected in the convex pinnae with anti-NC16A IgE and examined 48h post-injection. (A) MMP-9 KO mice developed significantly less severe skin disease as compared to WT mice. n=8, bar 1 vs. bar 2, *p<0.05, paired t-test. (B) Routine histology revealed typical dermal-epidermal separation in the skin of WT mice, but not MMP-9 KO mice. (C) Indirect IF identified reduced numbers of eosinophils infiltrating the skin of MMP-9 KO mice compared to WT mice. Green, anti-MBP (eosinophils). Red, anti-BP180; Blue, anti-Col VII.

Figure 5:. Levels of eotaxin-1, eotaxin-2, and MMP-9 are increased in the blister fluids of BP patients.

(A). Levels of eotaxin-1, −2, and −3 were quantified in blister fluids from BP patients and suction blister fluids from normal human controls (NC) by ELISA. Significantly higher levels of eotaxin-1 and −2 were present in BP blister fluids as compared to NC suction blister fluids. BP was compared to NC for each of the evaluated chemokines, n=12 for BP and n=16 for NC. **p<0.01, paired t-test. (B). Levels of MMP-9 were quantified in the blister fluids of patients with BP and pemphigus vulgaris (PV) using an enzymatic assay. Significantly higher levels of MMP-9 activity were detected in the blister fluids of BP patients as compared to the blister fluids of PV patients. n=3/group, **p<0.01, paired t-test.