Chenyan Lin

Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome

Bullous impetigo due to Staphylococcus aureus is one of the most common bacterial infections of man, and its generalized form, staphylococcal scalded skin syndrome (SSSS), is a frequent manifestation of staphylococcal epidemics in neonatal nurseries. Both diseases are mediated by exfoliative toxins (ETs), which show exquisite pathologic specificity in blistering only the superficial epidermis. We show that these toxins act as serine proteases with extremely focused molecular specificity to cleave mouse and human desmoglein 1 (Dsg1) once after glutamic acid residue 381 between extracellular domains 3 and 4. Mutation of the predicted catalytically active serine to alanine completely inhibits cleavage. The mutated ETs bind specifically to Dsg1 by immunofluorescence colocalization and by coimmunoprecipitation. Thus, ETs, through specific recognition and proteolytic cleavage of one structurally critical peptide bond in an adhesion molecule, cause its dysfunction and allow S. aureus to spread under the stratum corneum, the main barrier of the skin, explaining how, although they circulate through the entire body in SSSS, they cause pathology only in the superficial epidermis.

Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display

Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.

Enzymatic and molecular characteristics of the efficiency and specificity of exfoliative toxin cleavage of desmoglein 1.

Exfoliative toxins (ETs) from Staphylococcus aureus blister the superficial epidermis by hydrolyzing a single peptide bond, Glu381–Gly382, located between extracellular domains 3 and 4 of desmoglein 1 (Dsg1). Enzyme activity is dependent on the calcium-stabilized structure of Dsg1. Here we further define the characteristics of this cleavage. Kinetic studies monitoring the cleavage of Dsg1 by ETA, ETB, and ETD demonstrated kcat/Km values of 2–6 × 104 m–1 s–1, suggesting very efficient proteolysis. Proteolysis by ETA was not efficiently inhibited by broad spectrum serine protease inhibitors, suggesting that the enzyme cleavage site may be inactive or inaccessible before specific binding to its substrate. Using truncated mutants of human Dsg1 and chimeric molecules between human Dsg1 and either human Dsg3 or canine Dsg1, we show that for cleavage, human-specific amino acids from Dsg1 are necessary in extracellular domain 3 upstream of the scissile bond. If these residues are canine rather than human, ETA binds, but does not cleave, canine Dsg1. These data suggest that the exquisite specificity and efficiency of ETA may depend on the enzymes binding upstream of the cleavage site with a very specific fit, like a key in a lock.

Persistence of Anti-Desmoglein 3 IgG+ B-Cell Clones in Pemphigus Patients Over Years

Pemphigus vulgaris (PV) is a prototypic tissue-specific autoantibody-mediated disease in which anti-desmoglein 3 (Dsg3) immunoglobulin G (IgG) autoantibodies cause life-threatening blistering. We characterized the autoimmune B-cell response over 14 patient-years in two patients with active and relapsing disease, then in one of these patients after long-term remission induced by multiple courses of rituximab (anti-CD20 antibody). Characterization of the anti-Dsg3 IgG+ repertoire by antibody phage display (APD) and PCR indicated that 6 clonal lines persisted in patient 1 (PV3) over 5.5 years, with only one new clone detected. Six clonal lines persisted in patient 2 (PV1) for 4 years, of which 5 persisted for another 4.5 years without any new clones detected. However, after long-term clinical and serologic remission, ~11 years after initial characterization, we could no longer detect any anti-Dsg3 clones in PV1 by APD. Similarly, in another PV patient, ~4.5 years after a course of rituximab that induced long-term remission, anti-Dsg3 B-cell clones were undetectable. These data suggest that in PV a given set of non-tolerant B-cell lineages causes autoimmune disease and that new sets do not frequently or continually escape tolerance. Therapy such as rituximab, aimed at eliminating these aberrant sets of lineages, may be effective for disease because new ones are unlikely to develop.

Proteomic Analysis of Pemphigus Autoantibodies Indicates a Larger, More Diverse, and More Dynamic Repertoire than Determined by B Cell Genetics

In autoantibody-mediated diseases such as pemphigus, serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here, we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than that found by genetic studies of B cells. In addition, many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies in individual patients over several years indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies and explain why similar overall autoantibody titers may give variable disease activity.

Targeted Delivery of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand to Keratinocytes with a Pemphigus mAb

We determined the feasibility of using an anti-desmoglein (Dsg) monoclonal antibody, Px44, to deliver a biologically active protein to keratinocytes. Recombinantly produced Px44-green fluorescent protein (GFP) injected into mice and skin organ culture delivered GFP to the cell surface of keratinocytes. We replaced GFP with tumor necrosis factor -related apoptosis-inducing ligand (TRAIL) to produce Px44TRAIL. We chose TRAIL as a biologic model because it inhibits activated lymphocytes and causes apoptosis of hyperproliferative keratinocytes, features of various skin diseases. Px44TRAIL formed a trimer, the biologically active form of TRAIL. Standard assays of TRAIL activity showed that Px44TRAIL caused apoptosis of Jurkat cells and inhibited interferon-γ production by activated CD4+ T cells. Enzyme-linked immunoassay with Px44TRAIL showed delivery of TRAIL to Dsg. Immunofluorescence with Px44TRAIL incubated on skin sections and cultured keratinocytes or injected into mouse skin, human organ culture or human xenografts detected TRAIL on keratinocytes. Px44TRAIL caused apoptosis of hyperproliferative, but not differentiating, cultured keratinocytes through binding to Dsg3. Foldon, a small trimerization domain, cloned into Px44TRAIL maintained its stability and biological activity at 37° for at least 48 hr. These data suggest that such targeted therapy is feasible and may be useful for hyperproliferative and inflamed skin diseases.

Normal human skin is superior to monkey esophagus substrate for detection of circulating BP180-NC16A-specific immunoglobulin G antibodies in bullous pemphigoid

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease. Two antigens have been identified as targets of circulating autoantibodies (autoAbs) – BP180 and BP230 – with BP180 being a critical transmembrane adhesion protein of basal keratinocytes of the epidermis. The noncollagenous domain 16A (NC16A) of BP180 is the immunodominant epitope in patients with BP, and anti‐BP180‐NC16A IgG antibodies (Abs) correlate to disease activity. Routine serological testing and follow‐up of BP relies on indirect immunofluorescence (IIF) of serum Abs, commonly performed on monkey oesophagus (ME), and/or enzyme‐linked immunosorbent assay (ELISA) testing on recombinantly produced fragments of BP180 and BP230 (BP180‐NC16A, BP230‐C/N).

Targeted Delivery of Tumor Necrosis Factor|[ndash]|Related Apoptosis-Inducing Ligand to Keratinocytes with a Pemphigus mAb

We determined the feasibility of using an anti-desmoglein (Dsg) monoclonal antibody, Px44, to deliver a biologically active protein to keratinocytes. Recombinantly produced Px44-green fluorescent protein (GFP) injected into mice and skin organ culture delivered GFP to the cell surface of keratinocytes. We replaced GFP with tumor necrosis factor -related apoptosis-inducing ligand (TRAIL) to produce Px44TRAIL. We chose TRAIL as a biologic model because it inhibits activated lymphocytes and causes apoptosis of hyperproliferative keratinocytes, features of various skin diseases. Px44TRAIL formed a trimer, the biologically active form of TRAIL. Standard assays of TRAIL activity showed that Px44TRAIL caused apoptosis of Jurkat cells and inhibited interferon-γ production by activated CD4+ T cells. Enzyme-linked immunoassay with Px44TRAIL showed delivery of TRAIL to Dsg. Immunofluorescence with Px44TRAIL incubated on skin sections and cultured keratinocytes or injected into mouse skin, human organ culture or human xenografts detected TRAIL on keratinocytes. Px44TRAIL caused apoptosis of hyperproliferative, but not differentiating, cultured keratinocytes through binding to Dsg3. Foldon, a small trimerization domain, cloned into Px44TRAIL maintained its stability and biological activity at 37° for at least 48 hr. These data suggest that such targeted therapy is feasible and may be useful for hyperproliferative and inflamed skin diseases.

Targeted delivery of tumor necrosis factor-related apoptosis-inducing ligand to keratinocytes with a pemphigus monoclonal antibody

We determined the feasibility of using an anti-desmoglein (Dsg) monoclonal antibody, Px44, to deliver a biologically active protein to keratinocytes. Recombinantly produced Px44-green fluorescent protein (GFP) injected into mice and skin organ culture delivered GFP to the cell surface of keratinocytes. We replaced GFP with tumor necrosis factor -related apoptosis-inducing ligand (TRAIL) to produce Px44TRAIL. We chose TRAIL as a biologic model because it inhibits activated lymphocytes and causes apoptosis of hyperproliferative keratinocytes, features of various skin diseases. Px44TRAIL formed a trimer, the biologically active form of TRAIL. Standard assays of TRAIL activity showed that Px44TRAIL caused apoptosis of Jurkat cells and inhibited interferon-γ production by activated CD4+ T cells. Enzyme-linked immunoassay with Px44TRAIL showed delivery of TRAIL to Dsg. Immunofluorescence with Px44TRAIL incubated on skin sections and cultured keratinocytes or injected into mouse skin, human organ culture or human xenografts detected TRAIL on keratinocytes. Px44TRAIL caused apoptosis of hyperproliferative, but not differentiating, cultured keratinocytes through binding to Dsg3. Foldon, a small trimerization domain, cloned into Px44TRAIL maintained its stability and biological activity at 37° for at least 48 hr. These data suggest that such targeted therapy is feasible and may be useful for hyperproliferative and inflamed skin diseases.