Inhibition of Toxic Epidermal Necrolysis by Blockade of CD95 with Human Intravenous Immunoglobulin

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Science  16 Oct 1998:
Vol. 282, Issue 5388, pp. 490-493
DOI: 10.1126/science.282.5388.490


Toxic epidermal necrolysis (TEN, Lyell's syndrome) is a severe adverse drug reaction in which keratinocytes die and large sections of epidermis separate from the dermis. Keratinocytes normally express the death receptor Fas (CD95); those from TEN patients were found to express lytically active Fas ligand (FasL). Antibodies present in pooled human intravenous immunoglobulins (IVIG) blocked Fas-mediated keratinocyte death in vitro. In a pilot study, 10 consecutive individuals with clinically and histologically confirmed TEN were treated with IVIG; disease progression was rapidly reversed and the outcome was favorable in all cases. Thus, Fas-FasL interactions are directly involved in the epidermal necrolysis of TEN, and IVIG may be an effective treatment.

Alterations in the control of apoptosis, a type of cell death, are involved in the pathogenesis of several human diseases (1, 2). Apoptosis can be triggered by interaction between a cell-surface death receptor such as Fas and its respective ligand (Fas ligand: FasL or CD95L).

TEN (or Lyell's syndrome) is a severe drug-induced skin disease in which apoptotic epidermal cell death results in the separation of large areas of skin at the dermo-epidermal junction (Fig. 1A), producing the appearance of scalded skin (3, 4). TEN occurs at an estimated incidence of 0.4 to 1.2 cases per million, most frequently as a result of sulfonamide, anticonvulsant, or nonsteroidal anti-inflammatory drug use, and is associated with a mortality rate of about 30% (3, 5). There is no known effective treatment for TEN. Keratinocyte apoptosis is rare in the normal epidermis, but is abnormally increased during TEN (3, 4). The mechanisms responsible for enhanced keratinocyte apoptosis in TEN remain unclear (4).

Figure 1

(A) Detachment of the epidermis (star) from the dermis as a result of extensive keratinocyte death in a skin sample from an individual with TEN. (B) Detection of sFasL in serum samples from patients with TEN (n = 5), MPR (n = 5), and healthy controls (n = 5), using an sFasL ELISA (25).

We screened serum samples from patients with clinically and histologically confirmed TEN (6), extensive drug-induced maculo-papular rash (MPR), and healthy controls for soluble FasL (sFasL) content (7, 8). Using a specific enzyme-linked immunosorbent assay (ELISA), we detected high concentrations of sFasL in the sera of patients with TEN, whereas sFasL was virtually undetected in patients with MPR or in healthy controls (Fig. 1B).

Keratinocyte apoptosis, which precedes epidermal detachment, is an early morphologic feature of TEN (4). Therefore, we investigated if the sFasL detected in the sera of patients with TEN resulted from cleavage of membrane-bound FasL produced in their epidermis (7, 9, 10). Fas signaling is known to be functional in human keratinocytes in vitro (11). Under basal conditions, keratinocytes express little FasL, and it is nonlytic in vitro (12–14). Following appropriate stimulation however, keratinocyte FasL can be induced to become lytic (12). To assess the possible involvement of Fas and its ligand in the process of keratinocyte cell death that leads to TEN, we analyzed Fas and FasL expression in skin samples from individuals with TEN (n = 7), MPR (n = 4), and in healthy controls (n = 5). Skin sections from individuals with TEN showed keratinocyte apoptosis (Fig. 2A), and immunohistochemical analysis of adjacent frozen skin sections with monoclonal antibody (mAb) A11 to FasL (Fig. 2B) revealed that keratinocytes in all the TEN samples, but not control or MPR samples, expressed large amounts of FasL, whereas Fas expression remained unchanged compared to controls (Fig. 2C).

Figure 2

Up-regulated keratinocyte FasL expression in patients with TEN induces Fas-mediated target cell death. (A) Histological analysis of a skin section from a patient with TEN [patient BG (Table 1)]. Arrows, apoptotic keratinocytes. Bar, 35 μm. (B) Immunoblot of mAb A11 to FasL. Lane 1, 293T-mock–transfected cell lysate; lane 2, 293T-FasL–transfected cell lysate. (C) Immunohistochemical analysis of skin sections from a representative healthy control and a TEN patient exposed to anti-Fas, anti-FasL, or isotype control antibodies (26). Bar, 35 μm. (D) Flow cytometric analysis of apoptosis (annexin-FITC) in Fas-sensitive Jurkat cells overlaid for 6 hours on skin cryosections from healthy controls, patients with MPR, or TEN in the absence or presence of FasL-blocking antibody (NOK1) (27). Results of a representative experiment performed in duplicate (error bars) in three to five separate individuals (five controls, three MPR, three TEN). (E) Susceptibility of Jurkat cells and primary human epidermal keratinocytes (HEK) to rhsFasL. Cells were incubated with FLAG-tagged sFasL (rhsFasL, Alexis Corp.) plus anti-FLAG, [enhancer (1 μg/ml), Alexis Corp.] for 6 and 16 hours, respectively. Viability was determined using a cell proliferation assay (WST-1, Boehringer GmbH, Mannheim, Germany).

To determine if keratinocyte FasL in patients with TEN could induce Fas-mediated cell death, we examined the lytic capacity of cutaneous FasL in vitro. Frozen skin sections from healthy controls (n = 5) and patients with MPR (n = 3) or TEN (n = 3) were overlaid with Fas-sensitive Jurkat cells for 6 hours, and Jurkat cell apoptosis was subsequently assessed using an annexin–fluorescein isothiocyanate (FITC) (Fig. 2D) or cytochrome c assay (15). With both assays, TEN skin sections were reproducibly found to induce three- to fourfold more target cell death than did skin sections from healthy controls or patients with MPR (Fig. 2D). The cytotoxicity induced by TEN skin sections was completely abrogated by incubation with FasL blocking mAb (Fig. 2D) or by the addition of Fas-Fc, which shows that keratinocyte FasL in TEN can induce Fas-mediated apoptosis. Human keratinocytes were also sensitive to recombinant human sFasL (rhsFasL) (Fig. 2E). These results suggest a role for keratinocyte FasL in the keratinocyte apoptosis observed in TEN.

IVIG is a blood product prepared from the pooled plasma of healthy donors. IVIG can modulate cytokine release, induce functional blockade of certain receptors, and is increasingly used to treat inflammatory and autoimmune diseases (16). We investigated whether IVIG could interfere with Fas-mediated keratinocyte cell death by incubating them with IVIG before rhsFasL exposure. Using concentrations of rhsFasL that induce 75% keratinocyte apoptosis, IVIG (30 mg/ml; calculated equivalent of the daily dose used for treating a 60-kg individual) completely inhibited Fas-mediated keratinocyte apoptosis (Fig. 3A). Protection from rhsFasL was also observed when human keratinocyte, hepatocarcinoma or lymphoblastoid cell lines were preincubated with IVIG (Fig. 3A), showing that the protective effect of IVIG is not limited to keratinocytes. The effect of IVIG was specific, because similar concentrations of albumin had no effect (Fig. 3A). In contrast, preincubation of rhsFasL with IVIG did not inhibit its lytic capacity (17), showing that IVIG inhibits Fas-mediated cell death by blocking the Fas receptor rather than interacting with FasL. The inhibition of Fas-FasL interaction by IVIG was further confirmed by using an ELISA in which receptor-ligand interaction was measured in the presence or absence of IVIG. Binding of rhsFasL to Fas-Fc, but not binding of rhsTRAIL [recombinant human soluble TRAIL (TNF-related apoptosis-inducing ligand)] to TRAILR2-Fc (control), was significantly inhibited by IVIG (Fig. 3B).

Figure 3

Inhibition of Fas-mediated keratinocyte apoptosis by antibody to Fas contained in IVIG. (A) Effect of preincubation of primary human keratinocytes (HEK), or the human keratinocyte (HaCaT), hepatocarcinoma (HepG2), Fas-sensitive (A20), and resistant (A20R) lymphoblastoid cell lines with IVIG, vehicle (no IVIG), or albumin on the susceptibility to rhsFasL (28). Viability is the percent of the viability observed in the absence of rhsFasL. Shown are results of a representative experiment performed twice separately in triplicate (error bars). (B) Effect of IVIG on Fas-FasL (left) and TRAIL-TRAILR2 (right) interaction. Microtiter plates coated with Fas-Fc or TRAILR2-Fc fusion proteins were incubated with rhsFasL or rhsTRAIL in the presence or absence of IVIG (30 mg/ml), and ligand-receptor binding was measured as described (10, 24). OD, optical density measured at 490 nm wavelength. (C) Immunodetection of Fas-comp (Fas), but not related TNFR1-comp (TNFR1) or albumin (Alb.), with IVIG. Left, IVIG staining; middle, anti-Fas staining; right, ponceau staining (29). (D) Susceptibility of A20 cells to rhsFasL after preincubation with vehicle, IVIG, or Fas-Fc or TRAIL-R2-Fc (as control) immunoabsorbed IVIG. Viability was determined as previously described. Results of a representative experiment performed twice in triplicate (error bars).

To determine if the Fas inhibitory activity of IVIG is due to the presence of naturally occuring anti-Fas immunoglobulin (Ig), we determined whether IVIG bound to human Fas, and whether depletion of anti-Fas Ig from IVIG abrogated their capacity to inhibit Fas-mediated apoptosis. Immunodetection with IVIG of albumin or purified recombinant protein constructs containing the extracellular domains of human Fas (Fas-comp) or tumor necrosis factor receptor (TNFR1-comp) fused to a 55–amino acid linker [comp: cartilage oligomeric matrix protein pentamerization domain (18)] showed that IVIG bound to Fas-comp (Fig. 3C), not to albumin, and weakly to TNFR1-comp, suggesting that there may also be some anti-TNFR1 activity in IVIG. Depletion of anti-Fas Ig from IVIG by several passages on a Fas-Fc affinity column specifically abrogated the ability of IVIG to bind Fas-comp and block Fas-mediated cell death (Fig. 3D).

In an open, noncontrolled pilot study, 10 consecutive patients with TEN were treated in three clinical centers (Geneva, Lausanne, and Bern University Hospitals) with IVIG at doses ranging from 0.2 to 0.75 g/kg of body weight per day for four consecutive days. In all 10 patients, the progression of skin disease was rapidly interrupted after IVIG infusion (within 24 to 48 hours), which was accompanied by rapid skin healing and a favorable vital outcome without significant adverse effects (Table 1). Confirmation of the efficacy of IVIG in the treatment of TEN will require a controlled multicentric clinical trial.

Table 1

Clinical characteristics and evolution of 10 consecutive patients with TEN treated by IVIG (6).

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Our results suggest that up-regulation of keratinocyte FasL expression is the critical trigger for keratinocyte destruction during TEN. The observed therapeutic effect of IVIG in patients with TEN is likely to involve inhibition of Fas-mediated keratinocyte death by naturally occurring Fas-blocking antibodies contained within IVIG preparations. IVIG may also prove useful in the treatment of other diseases that are due to Fas-mediated tissue destruction, including graft-versus-host disease, Hashimoto's thyroiditis, and fulminant hepatitis (2,19).

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: french{at}


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