Passive transfer of anti-laminin 5 antibodies induces subepidermal blisters in neonatal mice.(1)
Laminin 5 (a3b3g2) is a heterotrimeric adhesion molecule that is associated with anchoring filaments in the lamina lucida of human epidermal basement membrane (BM). Laminin 5 is produced by human keratinocytes (HK). In vitro, HK deposit laminin 5 in their extracellular matrix (ECM) where this protein serves as the major integrin ligand for these cells. Interestingly, certain monoclonal anti–laminin 5 antibodies have been shown to impair HK adhesion to ECM in vitro.
Laminin 5 was isolated from the ECM of cultured HK as described previously. Confluent cultures of HK were sequentially extracted with 1% Triton X-100 in PBS, 2 M urea in 1 M NaCl, and 8 M urea to yield culture dishes coated with laminin 5. Urea-insoluble proteins were detached from the surface of culture dishes with a disposable cell scrapper and suspended in 0.5% (wt/vol) SDS; all extraction buffers contained 1 mM phenylmethylsulfonyl fluoride and 2 mM N-ethylmaleimide. Laminin 5 derived from HK ECM was dialyzed against 0.1% SDS in Tris-buffered saline at 4C, concentrated by solvent recovery (Sephadex G-75), and stored at -70C.
The laminin 5 purified from HK ECM was recovered as a complex of five disulfide-linked polypeptides corresponding to the unprocessed and processed a subunits (200 and 165 kDs, respectively), the 140 kD b subunit, and the unprocessed and processed g subunits (155 and 105 kDs, respectively). Anti–laminin 5 antisera did not bind BMs in human or murine blood vessels (a tissue site of laminin 1 [a1b1g1]), nerves (synapses containing laminin 3 [a1b2g1), or kidney (a site of laminins 1 and 3), BMs in human gall bladder or syno- vium, the basal lamina of striated muscle in murine tongue (a site of laminin 2 [a2b1g1]), or any BMs in rabbit skin. IgG in immune and positive control rabbit sera (but not preimmune or normal rabbit sera) specifically iden- tified polypeptides that correspond to the previously described unprocessed and/or processed a, b, and g subunits of laminin 5 in total protein extracts of cultured HKs and BALB/c epider- mal cells as well as HK ECM. Anti–laminin 5 (but not preimmune, control) sera specifically bound laminin 5 in extracts of HK ECM, but showed no reactivity to collagen types I, III, IV, V, or VII, laminins 1 or 2, human plasma fibronectin, or human as well as bovine serum albumin.
Neonatal BALB/c mice (24–36 h of age) were injected subcutaneously (along the back) or intraperito- neally with purified rabbit anti–laminin 5 (or normal rabbit) IgG daily for 2–4 d. Within 24–48 h, mice that received total doses of anti–laminin 5 IgG exceeding 5 mg/g body wt (n = 17) developed tense blisters on their abdomen, feet, and/or ears. Blisters were not localized to injection sites, but rather predominated in areas exposed to friction or trauma.
Time course studies after passive transfer of 5 mg/g body wt of anti–laminin 5 IgG to neonatal BALB/c mice indicated that while no clinical or histologic alterations were evident within 3 h microscopic subepidermal blisters were detectable at 6 h, epidermal peeling developed within 12 h, and frank, tense blisters were present within 24–48 h after the administration of specific immunoglobulin. Interestingly, BALB/c mice that received > 5 mg/g body wt of anti–laminin 5 IgG continued to develop new blisters as late as 8 d after passive transfer of antibody. Blisters at these and earlier time points were replaced by erosions and/or crusts that were followed within a few days by reepithelization. To deposits of rabbit IgG, continuous deposits of murine C3 were also found in the epidermal BMs of all neonatal BALB/c mice that received anti–laminin 5 IgG. Ti- ters of circulating rabbit anti–BM IgG in these mice correlated with the dose of passively transferred antibody and ranged from 5,120 to 40,960 in mice receiving total doses of 1–20 mg/g body wt of anti–laminin 5 IgG. Received 5 mg/g body wt of anti–laminin 5 IgG found that circulating anti–BM antibodies were present in their circulation for as long as 4 wk
