Elevated expression and release of tissue-type, but not urokinase-type, plasminogen activator after binding of autoantibodies to bullous pemphigoid antigen 180 in cultured human keratinocytes

Abstract

In bullous pemphigoid (BP), the binding of BP180-specific antibodies to their hemidesmosomal target antigen is not sufficient for blister formation, but must be accompanied by the release of proteases. Using plasminogen activator (PA) knock-out mice, the PA system has previously been shown to be a prerequisite for blister formation in experimental murine BP. Here, we found elevated levels of plasmin and tPA, but not of uPA, in blister fluid from BP patients (n = 7) compared to blisters from patients with toxic epidermal necrolysis (n = 4) and suction blisters in healthy controls (n = 7). Subsequently, we addressed the question whether keratinocytes release PA in response to the binding of anti-BP180 antibodies. Treatment of cultured normal human keratinocytes with BP IgG, but not with control IgG, led to both increased protein and mRNA levels of tPA, but not of uPA, as determined by ELISA and RT-PCR, respectively. The specificity of this finding was confirmed using BP180-deficient keratinocytes from a patient with generalized atrophic benign epidermolysis bullosa, where no tPA release was observed after stimulation with BP IgG. Our results show the elevated expression and release of tPA from normal human keratinocytes upon stimulation with antibodies to human BP180. Keratinocytes, by secreting tPA, may thus play an active role in blister formation of BP.

Fig. 1

Elevated levels of plasmin and tPA were present in blister fluid of patients with bullous pemphigoid compared to both suction blisters in healthy controls and blisters of patients with toxic epidermal necrolysis. Blister fluid from patients with bullous pemphigoid (BP; n = 7; □), from suction blisters (SB) raised on the forearm of healthy volunteers (n = 7; ), and from blisters in patients with toxic epidermal necrolysis (TEN; n = 4; ▪) were analysed for reactivity with (a) tPA, (b) uPA and (c) plasmin in duplicate by ELISA and chromogenic assay, respectively. Bars show mean ± SD (ng/ml for tPA and uPA, µg/ml for plasmin levels). Asterisks indicate statistical significance between levels in BP blister fluid compared to levels in both blister fluid of SB in healthy volunteers and of blisters in TEN patients (*P < 0·05, **P >  0·01, ***P < 0·001).

Fig. 2

Cultured normal human epidermal keratinocytes (NHEK) released tPA, but not uPA, in a time-dependent fashion after incubation with BP IgG. NHEK were incubated with 4 mg/ml IgG affinity-purified from the serum of a patient with bullous pemphigoid (BP-1 IgG; □), from a healthy control (normal IgG; ), and with medium alone (▪), respectively. After incubation times of 3 h, 6 h, 9 h, 12 h, and 24 h, culture supernatants were analysed for (a) tPA and (b) uPA reactivity in quadruplicate by ELISA. Bars show mean ± SD (ng/ml). Asterisks indicate a statistically significant difference between BP IgG- and normal IgG-treated cells (*P < 0·05, **P >  0·01, ***P < 0·001). This pattern is representative of the pattern seen in 2 separate experiments with keratinocytes from different donors.

Fig. 3

Cultured normal human epidermal keratinocytes (NHEK) released tPA but not uPA in response to IgG purified from the serum of patients with bullous pemphigoid. Cultured NHEK were incubated with 4 mg/ml IgG from BP patients (BP-1, BP-2, BP-3; □), healthy controls (normal human-1, normal human-2; ), and with medium alone (▪) for 9 h. Levels of (a) tPA and (b) uPA in the culture supernatants were analysed in quadruplicate by ELISA. Whereas tPA levels in response to BP IgG were about 3-fold higher compared to treatment with normal human IgG, uPA levels, although about 10-fold higher than tPA levels, were not different between the 2 groups. Bars indicate mean ± SD (ng/ml). Asterisks indicate a statistically significant difference between BP IgG- and normal IgG-treated cells (P < 0·001). This pattern is representative of the pattern seen in 2 separate experiments with keratinocytes from different donors.

Fig. 4

The tPA release from cultured human keratinocytes, upon incubation with BP IgG, was mediated by antibodies to BP180. BP180-deficient GABEB keratinocytes were treated for 9 h with 4 mg/ml IgG affinity-purified from 3 bp patients (BP-1, BP-2, BP-3; □), 2 healthy volunteers (controls (normal human-1, normal human-2) ), medium alone (▪), and known inducers of tPA, including IL-1β, TNFα, and normal human serum (NHS; ). tPA levels were analysed in quadruplicate by ELISA. Bars show mean ± SD (ng/ml).

Fig. 5

Antibodies to BP180 induced both elevated tPA protein and mRNA levels in cultured normal human epidermal keratinocytes (NHEK). Cultured NHEK were treated for a 9 h incubation time with 4 and 8 mg/ml IgG from rabbit R594 (immunized against recombinant human BP180 NC16A; □), with preimmune rabbit IgG (), and with medium alone (▪). (a) Culture supernatants were assayed for tPA and uPA reactivity in quadruplicate by ELISA. Bars indicate mean ± SD (ng/ml). (b) tPA mRNA was detected by RT-PCR and expressed as ratio to GAPDH. Asterisks indicate statistical significant difference between R594 IgG- and preimmune IgG-treated cells (*P < 0·05, ***P < 0·001). This pattern is representative of the pattern seen in 2 separate experiments with keratinocytes from different donors.