Pemphigus Vulgaris and Pemphigus Foliaceus Antibodies are Pathogenic in Plasminogen Activator Knockout Mice

Pemphigus Vulgaris and Pemphigus Foliaceus Antibodies are Pathogenic in Plasminogen Activator Knockout Mice My G. Mahoney,1 Zhi Hong Wang, and John R. Stanley Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

Previous studies have suggested that urokinase plasminogen activator is required for blister formation in pemphigus vulgaris and pemphigus foliaceus. Other studies, however, have shown that downregulation of plasminogen activator does not inhibit blisters induced by pemphigus immunoglobulin G. To eliminate the possibility that small amounts of urokinase plasminogen activator might be sufficient for blister formation, we passively transferred pemphigus immunoglobulin G to urokinase plasminogen activator knockout neonatal mice. Pemphigus foliaceus and pemphigus vulgaris immunoglobulin G caused gross blisters and acantholysis in the superficial and suprabasal epidermis, respectively, to the same degree in knockout and control mice, demonstrating that urokinase plasminogen activator is not absolutely required for antibody-induced blisters. Some studies have shown elevated tissue-type plasminogen activator in pemphigus lesions. Tissue-type plasminogen

activator, however, is not necessary for blister formation, because pemphigus foliaceus and pemphigus vulgaris immunoglobulin G caused blisters to the same degree in tissue-type plasminogen activator knockout and control mice. To rule out that one plasminogen activator might compensate for the other in the knockout mice, we bred urokinase plasminogen activator, tissue-type plasminogen activator double knockouts. After passive transfer of pemphigus foliaceus and pemphigus vulgaris immunoglobulin G these mice blistered to the same degree as the single knockout and control mice, and histology indicated blisters at the expected level of the epidermis. These data definitively demonstrate that plasminogen activator is not necessary for pemphigus immunoglobulin G to induce acantholysis in the neonatal mouse model of pemphigus. Key words: cell adhesion/desmoglein/desmosome/protease. J Invest Dermatol 113:22–25, 1999

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There were also studies, however, that suggested that blister formation in pemphigus is indirectly mediated by release of proteases secondary to antibody binding (Schiltz et al, 1979; Farb et al, 1978). Furthermore, under some experimental conditions protease inhibitors were able to inhibit acantholysis induced by PV antibodies added to human epidermal culture and passively transferred to neonatal mice (Farb et al, 1978; Naito et al, 1989). Most studies that have attempted to identify the specific proteases involved in the pathophysiology of pemphigus have implicated plasminogen activator (PA). PF and PV immunoglobulin (Ig) G added to human keratinocyte cell culture caused PA release, mostly of the urokinase type (uPA) (Hashimoto et al, 1983). These same authors showed that plasminogen (the substrate for PA) enhanced the ability of pemphigus IgG to cause acantholysis in organ culture (Hashimoto et al, 1983). Further studies of human skin organ culture demonstrated that a rabbit anti-uPA antibody and PA inhibitor 2 (a potent inhibitor of uPA) blocked acantholysis induced by PV and PF IgG (Morioka et al, 1987; Hashimoto et al, 1989). Finally, recent studies point to a potential mechanism of antibody-mediated PA release from keratinocytes through the activation of protein kinase C (Esaki et al, 1995). In spite of these studies there are problems with implicating PA in pemphigus blister formation. Studies showing PA release by human keratinocytes are mostly based on cell culture, whereas acantholysis and inhibition of acantholysis is mostly demonstrated in organ culture. Although uPA has been implicated in cell culture, tissue type PA (tPA) increases have been found in lesional skin of pemphigus (Jensen, 1988; Baird, 1990). Furthermore, PA is elevated

n the two major forms of pemphigus, pemphigus vulgaris (PV) and pemphigus foliaceus (PF), autoantibodies against desmogleins cause loss of keratinocyte cell adhesion (also termed acantholysis) and blister formation (Stanley, 1989, 1990). The autoantibodies from PF patients are directed against desmoglein (Dsg) 1 and cause blisters in the superficial living epidermis, whereas the autoantibodies in PV are directed against Dsg3 (with or without coexisting anti-Dsg1 antibodies) and cause blisters in the deep epidermis, just above the basal layer (i.e., suprabasilar acantholysis) (Buxton et al, 1993; Stanley, 1993). Desmogleins are transmembrane glycoproteins of desmosomes, which are cell–cell adhesion structures. Therefore, when it was discovered that pemphigus autoantibodies bind desmogleins, it was postulated that these antibodies might directly interfere with an adhesion function of these desmosomal molecules (Koulu et al, 1984; Amagai et al, 1991). Manuscript received January 21, 1999; revised March 17, 1999; accepted for publication March 25, 1999. Reprint requests to: Dr. John R. Stanley, Department of Dermatology, University of Pennsylvania, 211 CRB, 415 Curie Blvd., Philadelphia, PA 19104, U.S.A. 1Current address: M.G. Mahoney, Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107, U.S.A. Abbreviations: PA, plasminogen activator; PF, pemphigus foliaceus; PV, pemphigus vulgaris; tPA, tissue-type plasminogen activator; uPA, urokinasetype plasminogen activator.

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in the lesional skin of many other skin diseases that are not characterized by acantholysis (Jensen, 1988; Baird, 1990; Spiers et al, 1994, 1996). Finally, in the neonatal mouse model of pemphigus, dexamethasone markedly suppressed (but did not completely eliminate) PA activity induced by pemphigus IgG, but did not affect blister formation (Anhalt et al, 1986). Therefore, although some studies suggest that PA, particularly uPA, may be critical to blister formation in pemphigus, other studies do not support this hypothesis. In this study we attempt to answer, definitively, the question of whether PA expression is necessary for PF and PV antibodies to induce the characteristic pathology in these diseases by using passive transfer of pemphigus IgG to neonatal mice with targeted disruptions in the PA genes. MATERIALS AND METHODS PA knockout mice We purchased mice with a targeted mutation in uPA and tPA, as reported by Carmeliet et al (1994), and bred back to a C57Bl/6 background (Jackson Laboratories, Bar Harbor, ME). These mice had the following genotypes: uPA–/–tPA1/1 and uPA1/1tPA–/–. In addition to crossing mice of identical genotypes to propagate uPA–/– and tPA–/– mice, we intercrossed uPA–/– tPA1/1 3 uPA1/1tPA–/– then backcrossed to obtain the following breeding pairs: (i) tPA–/–uPA1/– 3 tPA1/– uPA–/–; (ii) tPA1/–uPA1/– 3 tPA–/–uPA1/–; and (iii) tPA1/–uPA1/– 3 tPA1/–uPA–/–. These breedings supplied tPA–/– and uPA–/– mice as well as tPA–/–uPA–/– double knockouts.

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Figure 1. Southern blots of EcoRI restricted mouse tail DNA to identify uPA–/–, tPA–/–, and uPA–/–tPA–/– mice. (A) uPA probe d identifies a 5.7 kb wild-type allele (open arrowhead) and a 10 kb mutant allele (closed arrowhead). M1 shows DNA from a uPA–/– mouse; lane C shows DNA from a control uPA1/– mouse used to show both alleles. (B) tPA probe d labels a 5.5 kb wild-type allele (open arrowhead) and a 9.7 kb mutant allele (closed arrowhead) in DNA from a control tPA1/– mouse (lane C). M2 shows DNA from a tPA–/– mouse. (C) Lanes labeled M3 show DNA from a double knockout mouse with only mutant (closed arrowhead) uPA and tPA alleles, but no wild-type alleles (open arrowheads).

Genotyping of PA knockout mice Genomic DNA was extracted from the tails of mice and processed for EcoRI digestion and Southern blotting, as previously reported (Koch et al, 1997). A 1 kb Hind III–EcoRI probe from the 39 flanking region of the tPA gene and an 0.24 kb AccI– HindIII probe from the 39 flanking region of the uPA gene were generously provided by Peter Carmeliet, and are referred to as tPA probe ‘‘d’’ and uPA probe ‘‘d’’ as described (Carmeliet et al, 1994). These probes were used as templates with random primers to synthesize 32P-labelled probes for use on Southern blots, as previously reported (Koch et al, 1997). Neonatal mouse model of pemphigus Neonatal mice with varying uPA and tPA genotypes were injected with IgG purified from PV and PF sera, as originally described by Anhalt et al (1982). Litters of 1–2 d old neonates were injected with IgG. After µ16 h the animals were evaluated for gross blisters, then killed for histology and to harvest DNA for genotyping. The IgG from two PF sera and two PV sera were used; IgG from both PF sera gave identical results as did IgG from both PV sera. Specific procedures for preparation of IgG, injection into neonatal mice, and evaluation of mice were essentially as recently described (Mahoney et al, 1999). Mice were evaluated grossly, before the genotype was determined, for spontaneous blister formation. Histology was graded semiquantitatively, without knowing the genotype, on the following scale: –, no blister; 11, blister at edge only; 21, localized blisters , 50% of specimen; 31, extensive blisters . 50% of specimen; 41, very extensive blisters . 75% of specimen.

RESULTS Determination of pemphigus IgG dose levels to induce extensive and more limited blisters in neonatal mice Using normal C57Bl/6 mice as well as control littermates of uPA and tPA knockout mice with at least one wild-type allele for both PA, we determined approximate doses of PF and PV IgG that caused extensive and limited blistering. These doses averaged 4.4 mg per g (‘‘high dose’’) and 1.1 mg per g (‘‘low dose’’) for PF IgG, and 8 mg per g (‘‘high dose’’) and 3 mg per g (‘‘low dose’’) for PV IgG. For the IgG derived from pemphigus sera in this study, as in our previous studies, PF IgG was consistently more effective at inducing blisters as compared with PV IgG. IgG from normal human sera did not induce gross or microscopic blisters at any dose. uPA is not necessary for induction of blisters by pemphigus IgG Previous studies, discussed above, have implicated uPA as necessary for blister formation in pemphigus. To determine if uPA is absolutely necessary for, or modulates, blister formation in the neonatal mouse model of pemphigus, we bred uPA–/– mice. Southern blots of these mice and littermates with uPA probe d (see Materials and Methods) on EcoRI restricted genomic DNA results

Figure 2. PA null neonatal mice injected with PF and PV IgG develop blisters. (A) uPA–/– mouse injected with PF IgG 0.7 mg per g. (B) uPA–/– mouse injected with PV IgG 10 mg per g. (C) uPA–/–tPA–/– mouse injected with PF IgG 0.6 mg per g. (D) uPA–/–tPA–/– mouse injected with PV IgG 3 mg per g. Arrow indicates flaccid blister.

in a 5.7 kb fragment from the wild-type allele and a 10 kb fragment from the recombinant (knockout) allele in these mice (Fig 1A). Passive transfer of PF IgG and PV IgG at both high and low doses resulted in gross and histologic blisters in these mice, to the same extent as uPA1/– and uPA1/1 mice (Figs 2A, B, 3A, B, and 4) Furthermore, the histology of lesional skin was typical for PF and PV; that is there was acantholysis in the granular layer with PF IgG injection (Fig 3A) and there was suprabasilar acantholysis with PV IgG injection (Fig 3B). tPA is not necessary for blister formation in pemphigus and does not compensate for lack of uPA Because some studies have demonstrated increased tPA, rather than uPA, in pemphigus lesions, we passively transferred PF and PV IgG to tPA–/– neonates. These mice were genotyped by southern blotting with tPA probe d which identified a 9.7 kb mutant allele and a 5.5 kb wild-type allele (Fig 1B). tPA null mice showed blisters that were histologically typical of PF and PV when their respective immunoglobulins were injected (Fig 3C, D). Although there may have been a slight tendency for less extensive blistering in tPA–/– neonates, there was significant overlap in the extent of blistering with control mice (Fig 4). Because the measurement of blistering was semiquantitative at best, we did not perform a statistical evaluation, but we can

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Figure 3. Epidermal histology of PA null mice injected with pemphigus IgG show superficial and suprabasilar acantholysis from PF and PV IgG, respectively. (A) uPA–/– mouse injected with PF IgG 0.7 mg per g. (B) uPA–/– mouse injected with PV IgG 3 mg per g. (C) tPA–/– mouse injected with PF IgG 1 mg per g. (D) tPA–/– mouse injected with PV IgG 3 mg per g. (E) uPA–/– tPA–/– mouse injected with PF IgG 0.6 mg per g. (F) uPA–/–tPA–/– mouse injected with PV IgG 3 mg per g.

conclude clearly that tPA is not necessary for the typical blistering of PV or PF. Finally, we wanted to rule out the possibility that in mice null for one of the PA the other does not compensate, allowing blister formation (i.e., redundancy in null animals). We therefore bred uPA–/–tPA–/– null mice (Fig 1C). In these PA null mice, PV and PF IgG caused blistering to the same degree as in control and uPA–/– or tPA–/– mice (Figs 2C, D, 3E, F, and 4). The histology of the blisters in these mice was indistinguishable from control or single PA knockout mice (Fig 3). DISCUSSION These data indicate that, in the neonatal mouse model of pemphigus, PA is not necessary for blister formation and does not, in a major way, modulate the extent or localization of blister formation. These studies provide further evidence that pemphigus autoantibodies cause loss of cell adhesion directly by interference with desmoglein function rather than indirectly by release of proteases. These results support several lines of evidence that point to a direct effect of pemphigus antibodies on desmoglein function. The recently described mouse with a null mutation in Dsg3 has a phenotype very similar to PV patients with suprabasilar blisters in oral mucous membranes and traumatized skin (Koch et al, 1997). This result demonstrates that the loss of function of Dsg3 results in a similar loss of adhesion of keratinocytes as do anti-Dsg3 antibodies, suggesting that these antibodies cause loss of function. Further evidence for a direct effect of pemphigus antibodies on desmoglein function and against the idea of protease release as a significant factor in blister pathogenesis comes from recent data supporting ‘‘the desmoglein compensation hypothesis’’ as an explanation for the localization of blisters in PF and PV (Mahoney et al, 1999). This hypothesis states that where Dsg3 and Dsg1 expression overlap in the epidermis antibodies against either one will not cause spontaneous blisters, but antibodies against both will do so. On the other hand where Dsg3 or Dsg1 is expressed alone, antibodies against that desmoglein will cause a blister. These data

Figure 4. Similar extent of histologic blistering in control and PA mutant mice. High dose: mean for PF IgG 4 mg per g; for PV 8 mg per g. Low dose: mean for PF IgG 1 mg per g; for PV 3 mg per g. 1, At least one wild-type allele each for uPA and tPA. u-, uPA–/– with at least one wild-type allele for tPA. t-, tPA–/– with at least one wild-type allele for uPA. –/–, no PA wild-type alleles. Extent of blistering graded by a semiquantitative scale as described in Materials and Methods.

are consistent with the idea that antibodies in pemphigus interfere with desmoglein function, and are not consistent with the idea that antibodies that bind to a desmoglein release a protease which causes the blister (because in the later case the second desmoglein would not be expected to compensate). Although formally we have not ruled out that PA play a major part in human, as opposed to mouse, pemphigus, we think it highly unlikely that pemphigus autoantibodies would cause identical histologic lesions in mice and humans, but by different mechanisms. Although it is possible that PA and perhaps other proteases modulate the extent of blister formation in pemphigus patients, the weight of the current evidence suggests that the major pathophysiologic

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mechanism in pemphigus is a direct effect of autoantibodies on the function of desmogleins and the desmosomes in which they are found.

We thank Dr. Peter Carmeliet for providing the uPA and tPA DNA probes. This work was supported by grants from the NIAMS of the NIH.

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pemphigus autoantibody stimulates plasminogen activator activity of human epidermal cells. J Exp Med 157:259–272, 1983 Hashimoto K, Wun TC, Baird J, Lazarus GS, Jensen PJ: Characterization of keratinocyte plasminogen activator inhibitors and demonstration of the prevention of pemphigus IgG-induced acantholysis by a purified plasminogen activator inhibitor. J Invest Dermatol 92:310–314, 1989 Jensen PJ: Epidermal plasminogen activator is abnormal in cutaneous lesions. J Invest Dermatol 90:777–782, 1988 Koch PJ, Mahoney MG, Ishikawa H, et al: Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris. J Cell Biol 137:1091– 1102, 1997 Koulu L, Kusumi A, Steinberg MS, Klaus Kovtun V, Stanley JR: Human autoantibodies against a desmosomal core protein in pemphigus foliaceus. J Exp Med 160:1509–1518, 1984 Mahoney MG, Wang Z, Rothenberger K, Koch PJ, Amagai M, Stanley JR: Explanation for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris. J Clin Invest 103:461–468, 1999 Morioka S, Lazarus GS, Jensen PJ: Involvement of urokinase-type plasminogen activator in acantholysis induced by pemphigus IgG. J Invest Dermatol 89:474– 477, 1987 Naito K, Morioka S, Nakajima S, Ogawa H: Proteinase inhibitors block formation of pemphigus acantholysis in experimental models of neonatal mice and skin explants: effects of synthetic and plasma proteinase inhibitors on pemphigus acantholysis. J Invest Dermatol 93:173–177, 1989 Schiltz JR, Michel B, Papay R: Appearance of ‘‘pemphigus acantholysis factor’’ in human skin cultured with pemphigus antibody. J Invest Dermatol 73:575– 581, 1979 Spiers EM, Lazarus GS, Lyons-Giordano B: Expression of plasminogen activator enzymes in psoriatic epidermis. J Invest Dermatol 102:333–338, 1994 Spiers EM, Lazarus GS, Lyons-Giordano B: Expression of plasminogen activators in basal cell carcinoma. J Pathol 178:290–296, 1996 Stanley JR: Pemphigus and pemphigoid as paradigms of organ-specific, autoantibodymediated diseases. J Clin Invest 83:1443–1448, 1989 Stanley JR: Pemphigus: skin failure mediated by autoantibodies. JAMA 264:1714– 1717, 1990 Stanley JR: Cell adhesion molecules as targets of autoantibodies in pemphigus and pemphigoid, bullous diseases due to defective epidermal cell adhesion. Adv Immunol 53:291–325, 1993