T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis

Abstract

Clinical and histologic similarities between various eczematous disorders point to a common efferent pathway. We demonstrate here that activated T cells infiltrating the skin in atopic dermatitis (AD) and allergic contact dermatitis (ACD) induce keratinocyte (KC) apoptosis. KCs normally express low levels of Fas receptor (FasR) that can be substantially enhanced by the presence of IFN-gamma. KCs are rendered susceptible to apoptosis by IFN-gamma when FasR numbers reach a threshold of approximately 40,000 per KC. Subsequently, KCs undergo apoptosis induced by anti-FasR mAb's, soluble Fas ligand, supernatants from activated T cells, or direct contact between T cells and KCs. Apoptotic KCs show typical DNA fragmentation and membrane phosphatidylserine expression. KC apoptosis was demonstrated in situ in lesional skin affected by AD, ACD, and patch tests. Using numerous cytokines and anti-cytokine neutralizing mAb's, we found no evidence that cytokines other than IFN-gamma participate in this process. In addition, apoptosis-inducing pathways other than FasR triggering were ruled out by blocking T cell-induced KC apoptosis by caspase inhibitors and soluble Fas-Fc protein. Responses of normal human skin and cultured skin equivalents to activated T cells demonstrated that KC apoptosis caused by skin-infiltrating T cells is a key event in the pathogenesis of eczematous dermatitis.

Figure 1

(a) Representative histologic findings of acute eczematous dermatitis. A dense subepidermal inflammatory infiltrate and marked epidermal acantholytic and spongiotic changes progress to vesicle formation. Hematoxylin/eosin staining. ×400. (b–d) Signs of KC injury after coculture with autologous T cells. Photomicrographs from 96-well plates with an inverted microscope equipped with phase contrast. ×200. (b) Intact monolayer of cultured third-passage primary human KCs. The KCs are relatively uniform in size and morphology. (c) Intact monolayer of KCs after 3 days in coculture with unstimulated autologous CD45RO⁺ T cells. (d) Partly destroyed monolayer of KCs after 3 days in coculture with autologous CD45RO⁺ T cells stimulated with anti-CD2, anti-CD3, and anti-CD28 mAb’s. (e and f) Induction of KC apoptosis in vitro. Identification of apoptotic nuclei with HOECHST staining. ×200. (e) Intact monolayer of KCs after 3 days of coculture in Transwell plates with unstimulated CD45RO⁺ T cells. (f) Induction of KC apoptosis after coculture in Transwell plates with stimulated CD45RO⁺ T cells. Note bright, condensed nuclei and nuclear fragmentation (arrows), signs of KC apoptosis.

Figure 2

(a) Expression of FasR mRNA by primary human KCs. Lane 1: unprotected template. Lane 2: after 8 hours of KC culture. Lane 3: after 24 hours of KC culture. Lane 4: after 16 hours of KC culture followed by 8 hours with 1.0 μg/mL anti-FasR mAb. Lane 5: after 16 hours of KC culture followed by 8 hours with diluted (50%) supernatant (sup.) from stimulated CD45RO⁺ T cells. Lane 6: after 16 hours of KC culture followed by 8 hours with 1.0 ng/mL IFN-γ. A representative result of three experiments is shown. (b) IFN-γ and Fas-induced KC apoptosis. KC viability was monitored by ethidium bromide exclusion and flow cytometry. Treatments shown are control (KCs alone), 1 μg/mL activating anti-FasR mAb, 10 ng/mL IFN-γ, and 1 μg/mL anti-FasR mAb added 1 day after starting incubation with 10 ng/mL IFN-γ. ^AP < 0.05. (c) IFN-γ–induced FasR counts exhibit a threshold for KC apoptosis. KCs were pretreated with the indicated doses of IFN-γ; 1 μg/mL anti-FasR mAb was added 1 day after starting incubation with IFN-γ. KC viability was assessed by ethidium bromide exclusion and flow cytometry at day 3. FasR count 1 day after starting incubation with indicated doses of IFN-γ. (d) CD45RO⁺ T cell–induced KC death is inhibited by blocking IFN-γ. KC viability was monitored by ethidium bromide exclusion and flow cytometry. In the flow cytometry setting, KCs and T cells are gated according to forward and side scatter. Both cell populations were therefore monitored separately. Coculture of primary human KCs and autologous unstimulated or stimulated (with anti-CD2, anti-CD3, and anti-CD28 mAb) CD45RO⁺ T cells. ^AP < 0.05. Inhibition of CD45RO⁺ T cell–induced KC death by 1 μg/mL blocking anti–IFN-γ receptor mAb and 20 μg/mL neutralizing anti–IFN-γ mAb. Results in b–d represent mean ± SD of triplicate cultures from three different experiments. Control, KCs alone.

Figure 3

Induction and regulation of KC apoptosis in KC–T cell cocultures. (a) Coculture of primary human KCs and stimulated autologous CD45RO⁺ T cells. After 3 days of coculture, cells were stained with annexin V and subjected to flow cytometry. With the indicated stimuli, the number of apoptotic KCs increases and the cells show an increase in annexin V binding (open histograms). Filled histograms show live KCs. The percentage of apoptotic KCs is indicated at upper right. Panel 1: KC apoptosis induced by coculture with CD45RO⁺ T cells stimulated with anti-CD2, anti-CD3, and anti-CD28 mAb’s. Panel 2: Isotype control mAb. Panel 3: Blocking anti–IFN-γ receptor mAb (1 μg/mL). Panel 4: Fas-Fc protein (1 μg/mL). Panel 5: The caspase inhibitor Z-VAD-FMK (50 μM). Panel 6: Z-VAD-FMK (100 μM). (b) Coculture of primary human KCs and stimulated autologous CD45RO⁺ T cells in Transwell plates. After 3 days in coculture, permeabilized KCs were stained with propidium iodide and subjected to flow cytometry. Filled histograms demonstrate control KCs containing diploid DNA. The percentage of apoptotic KCs with hypodiploid DNA (open histograms) is shown in the upper right corner. Panel 1: Primary human KCs alone were cultured in the lower well. Panel 2: KCs were pretreated with 10 ng/mL IFN-γ for 24 hours, and KC apoptosis was determined 3 days after 1 μg/mL activating anti-FasR mAb was added. Panel 3: KC apoptosis induced by Transwell coculture with stimulated CD45RO⁺ T cells. Panel 4: Inhibition of KC apoptosis induced by IFN-γ and anti-FasR mAb, with 1 μg/mL ZB4 blocking mAb. Panels 5 and 6: Inhibition of KC apoptosis induced by Transwell coculture with stimulated CD45RO⁺ T cells, with 1 μg/mL Fas-Fc protein (panel 5), and with 10 μg/mL neutralizing anti–IFN-γ mAb (panel 6). Results in a and b are representative of three experiments.

Figure 4

Induction of KC apoptosis by type 1 and type 2 T cells. (a) Type 1 but not type 2 T cells mediated KC death. Coculture of primary human KCs and heterologous differentiated type 1 and type 2 T cells. ^AP < 0.05. (b) Cytokine content of differentiated and stimulated type 2 T cells as determined by ELISA. CD4⁺ T helper 2 (Th2) and CD8⁺ T cytotoxic 2 (Tc2) cells were stimulated with anti-CD2, anti-CD3, and anti-CD28 mAb’s or with a combination of anti-CD2, anti-CD3, and anti-CD28 mAb’s and IL-12. ^AP < 0.05. (c) Coculture of primary human KCs and heterologous differentiated type 2 T cells. KC viability was measured by ethidium bromide exclusion and flow cytometry at day 3. Control, KCs alone. CD4⁺ Th2 cells and CD8⁺ Tc2 cells were stimulated with anti-CD2, anti-CD3, and anti-CD28 mAb’s or with a combination of anti-CD2, anti-CD3, and anti-CD28 mAb’s and IL-12. ^AP < 0.05. (d) Levels of soluble FasL in T-cell supernatants as determined by ELISA. CD4⁺ Th1/Th2 and CD8⁺ Tc1/Tc2 cells were stimulated with anti-CD2, anti-CD3, and anti-CD28 mAb’s or with a combination of anti-CD2, anti-CD3, and anti-CD28 mAb’s and IL-12. Results shown represent three (a, c) to five (b, d) experiments and are shown as mean ± SD from triplicate cultures.

Figure 5

Features of inflammation in AD. Immunohistologic staining with 3-amino-9-ethylcarbazole substrate and counterstaining with hematoxylin. (a) Subepidermal inflammatory infiltrate, consisting mainly of lymphocytes. ×100. (b) CD4⁺ cells infiltrating the spongiotic epidermis. ×200. (c) CD8⁺ cells. ×200. (d) FasL⁺ cells in the dermal cellular infiltrate. ×200. (e) Strong immunoreactivity of FasR in the basal epidermis; weaker staining in the T-cell infiltrate. ×100. (f) Detection of IFN-γ receptor on KCs and infiltrating T cells. ×200.

Figure 6

Demonstration of apoptotic KCs in AD, atopy patch tests, ACD, and in vitro induced eczematous dermatitis. Skin sections were subjected to TUNEL (a, b, e–g) or HOECHST staining (c, d) without counterstaining. (a) Healthy normal skin as a negative control (left); lesional skin of acute AD (right). Red condensed and partly fragmented nuclei indicate positive staining of apoptotic KCs (some of these are indicated by arrows). ×400. (b) Left: Acute ACD; 2+ patch test 48 hours after exposure to 5% nickel sulfate. Right: 2+ atopy patch test 72 hours after exposure to 10,000 protein nitrogen units of house dust mite. ×400. (c) Normal, healthy skin as a negative control. ×200. (d) Left: Lesional skin of acute AD. Right: Acute ACD. 2+ patch test 48 hours after exposure to 5% nickel sulfate. Apoptotic cells show condensed or fragmented nuclei (some of these are indicated by arrows). ×400. (e) Normal human skin after 3 days of in vitro culture with unstimulated CD45RO⁺ T cells. Left: No TUNEL-stained KCs are detectable. Right: Normal human skin after 3 days of in vitro culture exposed to stimulated CD45RO⁺ T cells. Features of spongiosis and numerous apoptotic KCs are detectable (arrows). ×200. (f) Cultured skin equivalent after 3 weeks of in vitro culture. Left: KC layers and the stratum corneum are visible (hematoxylin/eosin staining). Right: TUNEL staining. No TUNEL-stained nuclei are detectable. ×400. (g) Cultured skin equivalent after 3 days exposed to stimulated CD45RO⁺ T cells. There are signs of KC damage (left). With the TUNEL technique, several apoptotic nuclei are stained in the basal and suprabasal epidermis (arrows, right). Results shown in e–g are representative of two experiments with healthy skin and three experiments with cultured skin equivalents.