Ultraviolet-Radiation-Induced Keratinocyte Apoptosis in C1q-Deficient Mice
2001; Elsevier BV; Volume: 117; Issue: 1 Linguagem: Inglês
10.1046/j.0022-202x.2001.01381.x
ISSN1523-1747
AutoresMatthew C. Pickering, Susanne Fischer, Margarita Lewis, Mark Walport, Marina Botto, H. Terence Cook,
Tópico(s)T-cell and B-cell Immunology
ResumoExposure to ultraviolet B radiation is an important trigger of both systemic and cutaneous disease flares in individuals with systemic lupus erythematosus. More than 90% of individuals with homozygous C1q deficiency develop a systemic-lupus-erythematosus-like illness, which is typically associated with a severe photosensitive rash. Apoptotic, human keratinocytes have been shown in vitro to bind C1q, in the absence of antibody. These observations, together with the hypothesis that a major source of the autoantigens driving the immune response in systemic lupus erythematosus comes from apoptotic cells, led us to investigate the effects of murine C1q deficiency on ultraviolet-radiation-induced keratinocyte apoptosis in vivo. In this work, we demonstrated C1q binding to apoptotic murine keratinocytes in vitro and showed for the first time that C1q is also present on sunburn cells in vivo. In addition to C1q, we detected C3 deposition on sunburn cells in both wild-type and C1q-deficient mice, suggesting activation of the alternative pathway. Following acute ultraviolet exposure in vivo, no difference in the rate of clearance of sunburn cells was found in C1q-deficient mice from three different genetic backgrounds, compared with strain-matched wild-type controls. Furthermore, chronic ultraviolet exposure did not result in the production of autoantibodies or the development of glomerulonephritis. Our findings suggest that C1q does not play a critical role in the physiologic clearance of apoptotic murine keratinocytes in vivo. Exposure to ultraviolet B radiation is an important trigger of both systemic and cutaneous disease flares in individuals with systemic lupus erythematosus. More than 90% of individuals with homozygous C1q deficiency develop a systemic-lupus-erythematosus-like illness, which is typically associated with a severe photosensitive rash. Apoptotic, human keratinocytes have been shown in vitro to bind C1q, in the absence of antibody. These observations, together with the hypothesis that a major source of the autoantigens driving the immune response in systemic lupus erythematosus comes from apoptotic cells, led us to investigate the effects of murine C1q deficiency on ultraviolet-radiation-induced keratinocyte apoptosis in vivo. In this work, we demonstrated C1q binding to apoptotic murine keratinocytes in vitro and showed for the first time that C1q is also present on sunburn cells in vivo. In addition to C1q, we detected C3 deposition on sunburn cells in both wild-type and C1q-deficient mice, suggesting activation of the alternative pathway. Following acute ultraviolet exposure in vivo, no difference in the rate of clearance of sunburn cells was found in C1q-deficient mice from three different genetic backgrounds, compared with strain-matched wild-type controls. Furthermore, chronic ultraviolet exposure did not result in the production of autoantibodies or the development of glomerulonephritis. Our findings suggest that C1q does not play a critical role in the physiologic clearance of apoptotic murine keratinocytes in vivo. antinuclear antibody anti-double-stranded DNA antibody anti-single-stranded DNA antibody sunburn cell Hereditary deficiency of each of the classical pathway components of complement (C1q, C1r, C1s, C4, and C2) is associated with an increased susceptibility to systemic lupus erythematosus (SLE) (Pickering et al., 2000Pickering M.C. Botto M. Taylor P.R. Lachmann P.J. Walport M.J. Systemic lupus erythematosus, complement deficiency and apoptosis.Adv Immunol. 2000; 76: 227-324Crossref PubMed Scopus (402) Google Scholar). Both the severity of disease and strength of this association are greatest for homozygous C1q deficiency, which represents the strongest genetic susceptibility factor for the development of SLE that has been identified in humans. Thirty-nine of the 42 (93%) individuals with homozygous C1q deficiency reported to date have developed an SLE-like illness (Pickering et al., 2000Pickering M.C. Botto M. Taylor P.R. Lachmann P.J. Walport M.J. Systemic lupus erythematosus, complement deficiency and apoptosis.Adv Immunol. 2000; 76: 227-324Crossref PubMed Scopus (402) Google Scholar). It has been hypothesized that a major source of the autoantigens driving the immune response in SLE comes from apoptotic cells (Casciola-Rosen et al., 1994Casciola-Rosen L.A. Anhalt G. Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.J Exp Med. 1994; 179: 1317-1330Crossref PubMed Scopus (1489) Google Scholar,Casciola-Rosen et al., 1996Casciola-Rosen L. Rosen A. Petri M. Schlissel M. Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus.Proc Natl Acad Sci USA. 1996; 93: 1624-1629https://doi.org/10.1073/pnas.93.4.1624Crossref PubMed Scopus (429) Google Scholar). Apoptotic keratinocytes exhibit surface blebs that have been shown to contain many lupus autoantigens (e.g., nucleosomal DNA, Ro, La, Sm, and small nuclear ribonucleoproteins). In addition to being concentrated in the surface blebs, these autoantigens may also be altered by apoptotic-specific proteases allowing the exposure of potentially immunogenic cryptic epitopes (Casciola-Rosen et al., 1999Casciola-Rosen L. Andrade F. Ulanet D. Wong W.B. Rosen A. Cleavage by granzyme B is strongly predictive of autoantigen status: implications for initiation of autoimmunity.J Exp Med. 1999; 190: 815-826Crossref PubMed Scopus (413) Google Scholar). Furthermore, nonimmune mice injected intravenously with syngeneic apoptotic thymocytes have been shown to develop an autoantibody response (Mevorach et al., 1998aMevorach D. Zhou J.L. Song X. Elkon K.B. Systemic exposure to irradiated apoptotic cells induces autoantibody production.J Exp Med. 1998; 188: 387-392Crossref PubMed Scopus (483) Google Scholar). Several lines of evidence support a role for complement and, specifically, C1q in the physiologic clearance of apoptotic cells. Human keratinocytes bind C1q in the absence of antibody when rendered apoptotic by ultraviolet B (UVB) exposure in vitro (Korb and Ahearn, 1997Korb L.C. Ahearn J.M. C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited.J Immunol. 1997; 158: 4525-4528PubMed Google Scholar). The uptake of apoptotic human neutrophils and lymphocytes by human macrophages in vitro has been shown to be complement dependent (Mevorach et al., 1998bMevorach D. Mascarenhas J.O. Gershov D. Elkon K.B. Complement-dependent clearance of apoptotic cells by human macrophages.J Exp Med. 1998; 188: 2313-2320Crossref PubMed Scopus (546) Google Scholar). C1q-deficient mice develop proliferative glomerulonephritis characterized by the presence of an increased number of glomerular apoptotic bodies (Botto et al., 1998Botto M. Dell'Agnola C. Bygrave A.E. et al.Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies.Nat Genet. 1998; 19: 56-59Crossref PubMed Scopus (1200) Google Scholar). Finally, using an in vivo model of apoptotic cell clearance during sterile peritonitis, C1q-deficient mice have recently been shown to exhibit a defect in the phagocytic uptake of apoptotic cells, suggesting a specific role for C1q in this process (Taylor et al., 2000Taylor P.R. Carugati A. Fadok V.A. et al.A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo.J Exp Med. 2000; 192: 359-366Crossref PubMed Scopus (599) Google Scholar). Impairment of the physiologic removal of apoptotic cells may therefore explain the association between hereditary C1q deficiency and SLE. Exposure to UVB radiation is an important trigger of both systemic and cutaneous disease flares in individuals with SLE. In addition, approximately 80% of patients with homozygous C1q deficiency develop a severe photosensitive rash (Pickering et al., 2000Pickering M.C. Botto M. Taylor P.R. Lachmann P.J. Walport M.J. Systemic lupus erythematosus, complement deficiency and apoptosis.Adv Immunol. 2000; 76: 227-324Crossref PubMed Scopus (402) Google Scholar). These observations, together with the hypothesis that a major source of the autoantigens driving the immune response in SLE comes from apoptotic cells, led us to investigate the effects of murine C1q deficiency on UVR-induced keratinocyte apoptosis in vivo. We exposed C1q-deficient mice to acute UVR (270–350 nm) and quantified the degree of subsequent epidermal keratinocyte apoptosis. The effect of chronic UVR exposure was also investigated to test the hypothesis that chronic induction of sunburn cells (SBC) may trigger an autoimmune response in the absence of C1q. Finally, the binding of C1q to apoptotic murine keratinocytes was examined both in vitro and in vivo. Mice used in this study were 8–12-wk-old wild-type C57BL/6 or C1q-deficient (C1qa–/–) mice (Botto et al., 1998Botto M. Dell'Agnola C. Bygrave A.E. et al.Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies.Nat Genet. 1998; 19: 56-59Crossref PubMed Scopus (1200) Google Scholar) back-crossed onto the C57BL/6 strain for seven generations. In all the experiments controls were matched for age, sex, and strain. All animal work was conducted in accordance with institutional guidelines. Twenty-four hours before exposure to UVR, the dorsal surface of the mice was shaved using electric clippers. Mice were exposed to the UVR source in cages placed directly under four 100 W UV lamps (Philips TL12 lamps, spectral output 270–350 nm). The spectral emission of these bulbs has been previously published (Gibbs et al., 1995Gibbs N.K. Traynor N.J. MacKie R.M. Campbell I. Johnson B.E. Ferguson J. The phototumorigenic potential of broad-band (270–350 nm) and narrow-band (311–313 nm) phototherapy sources cannot be predicted by their edematogenic potential in hairless mouse skin.J Invest Dermatol. 1995; 104: 359-363Crossref PubMed Scopus (63) Google Scholar) and is predominantly UVB but includes moderate UVA and minimal UVC output. As the lamps were not filtered to specifically remove the UVA and minimal UVC output, the source is referred to as ultraviolet radiation (UVR). The cage floor was 15 cm from the UVR source and an internal wire mesh was placed 2.5 cm above the cage floor, which prevented the mice from reaching toward the source. The source to skin distance was 12.5 cm. The total dose of UVB radiation was monitored during each timed exposure period using an IL-400 A UVB photometer (International Light, Newbury Port, MA) positioned 12.5 cm from the UVR source. Therefore, all UVR doses measured in our experiments refer specifically to the dose of UVB radiation received. Using this experimental design, exposure periods of 1, 3, and 6 min equated to UVB doses of 130, 400, and 800 mJ per cm2, respectively. At various time points following exposure, mice were sacrificed and an elliptical dorsal skin biopsy was removed. The biopsies were divided and either placed in formalin for histologic analysis or immediately snap frozen in OCT embedding matrix (CellPath, Newtown, Wales, U.K.) for subsequent immunostaining procedures. Apoptotic keratinocytes, also termed SBC, were identified by their typical appearance on hematoxylin and eosin staining, i.e., shrunken cells with densely staining eosinophilic cytoplasm and a hyperchromatic condensed pyknotic nucleus (Woodcock and Magnus, 1976Woodcock A. Magnus I.A. The sunburn cell in mouse skin: preliminary quantitative studies on its production.Br J Dermatol. 1976; 95: 459-468Crossref PubMed Scopus (103) Google Scholar). Three skin sections per mouse, approximately 1 cm in length, were analyzed in a blinded fashion for each time point. The total length of epidermis was then measured using calibrated image analysis software (Image-Pro Plus; Media Cybernetics, MD) and the SBC count was expressed as number per centimeter of epidermis analyzed. Chronic exposure of mice to UVR was performed in an identical manner to that described for the acute exposure experiments. The first cohort comprised 19 C57BL/6 wild-type and 16 C1qa–/– female mice. Mice were shaved once weekly and exposed to 130 mJ per cm2 UVB radiation three times per week for a total exposure period of 6 wk. After 1, 2, 3, and 6 wk of UVR, groups of mice were sacrificed and serum and skin sections were examined. The second cohort comprised 17 C57BL/6 wild-type and 14 C1qa–/– male mice. Mice were shaved once weekly and 24 h later exposed to 400 mJ per cm2 UVB radiation. UVR exposure was performed weekly from the age of 8 wk for a total period of 7 mo. Tail bleeding for autoantibody analysis was performed at monthly intervals during the exposure period. All mice were sacrificed at the age of 9 mo. Primary murine keratinocyte cultures were established using a recently published protocol with slight modifications (Hager et al., 1999Hager B. Bickenbach J.R. Fleckman P. Long-term culture of murine epidermal keratinocytes.J Invest Dermatol. 1999; 112: 971-976https://doi.org/10.1046/j.1523-1747.1999.00605.xCrossref PubMed Scopus (105) Google Scholar). Newborn C57BL/6 mouse pups were sacrificed and rinsed in 70% ethanol; the trunk skin was removed by dissection and incubated in 50 ml of decontamination medium [phosphate-buffered saline (PBS) containing penicillin/streptomycin 5 mg per ml and fungizone 125 µg per ml] for 30 min. The skin was then incubated overnight at 4°C in 20 mg per ml dispase solution (Life Technologies, Paisley, U.K.). The epidermal sheets were separated from the dermis and placed in 20 ml of serum-free keratinocyte-specific medium (Life Technologies). The solution was firmly shaken 50 times and any remaining epidermal pieces were removed. Following centrifugation the cell pellet was resuspended in medium and plated onto glass coverslips at 105 cells per ml. Cultures were grown in serum-free keratinocyte-specific medium containing 0.06 mM CaCl2, bovine pituitary extract 25 µg per ml (BPE, Life Technologies), recombinant epidermal growth factor 0.1 ng per ml (rEGF, Life Technologies), penicillin/streptomycin 500 µg per ml, and fungizone 12.5 µg per ml. Cultures were washed on alternate days with PBS. Confluent keratinocyte cultures were present between days 7 and 10 and were visualized using Diff-Quik rapid staining kit (Dade Behring, Dudingen, Switzerland). This contains the fixative solution, fast green in methanol, and utilizes two staining solutions containing eosin G and thiazine dye. Keratinocytes were identified on morphologic criteria and by their positive staining on immunofluorescence using an antibody directed against mouse cytokeratin (polyclonal rabbit antimurine MK14 antibody; Babco, Cambridge, U.K.). Apoptosis was induced by exposure of cultures in PBS to UVR at a UVB dose of 3000 mJ per cm2 and incubating the cultures in standard RPMI medium (Life Technologies) without rEGF and BPE for 24 h, in place of the keratinocyte-specific medium. For detection of C1q binding to apoptotic keratinocytes, purified mouse C1q (a gift from Dr. Franz Petry) was added to the cultures immediately following UVB exposure, at a final concentration of 10 µg per ml. Both skin sections and murine keratinocyte cultures were stained for C1q in the following manner. The samples were fixed in acetone for 30 min. The primary antibody (polyclonal rabbit antimouse C1q, a gift from Professor Mohammed Daha) was diluted in 20% C1q-deficient mouse serum and incubated for 1 h at a dilution of 1:50 in PBS. Sections or cultures were then washed for 5 min in PBS. The secondary antibody was a fluorescein isothiocyanate (FITC) conjugated, γ-chain specific, monoclonal mouse antirabbit IgG antibody (Sigma, Poole, U.K.) diluted in 20% C1q-deficient mouse serum and incubated for 1 h at a dilution of 1:100 in PBS. After a further three 5 min washes, samples were mounted in Permafluor aqueous medium (Immunon, Pittsburgh, PA) and examined by fluorescent microscopy. C3 staining was performed by immunofluorescence using an FITC-conjugated goat antimouse C3 (ICN Cappell, Basingstoke, U.K.). FITC-conjugated goat IgG was used as a negative control (Sigma). The serum was stored at -70°C before analysis. Levels of antinuclear antibodies (ANA) were detected by indirect immunofluorescence using Hep-2 cells and anti-double-stranded DNA antibodies (antidsDNA) were detected by indirect immunofluorescence using Crithidia luciliae (The Binding Site, Birmingham, U.K.) as previously described (Mitchell et al., 1999Mitchell D.A. Taylor P.R. Cook H.T. Moss J. Bygrave A.E. Walport M.J. Botto M. Cutting edge: C1q protects against the development of glomerulonephritis independently of C3 activation.J Immunol. 1999; 162: 5676-5679PubMed Google Scholar). Serum samples were screened at dilutions of 1:80 (ANA) and 1:20 (antidsDNA) and positive samples were titrated to end-point. Antibodies to single-stranded DNA (antissDNA) were measured as previously described (Burlingame and Rubin, 1990Burlingame R.W. Rubin R.L. Subnucleosome structures as substrates in enzyme-linked immunosorbent assays.J Immunol Meth. 1990; 134: 187-199Crossref PubMed Scopus (108) Google Scholar). Samples were screened at a 1:50 dilution and the results were expressed as arbitrary ELISA units (AEU) relative to a standard positive sample (derived from an MRL.Mp.lpr/lpr mouse) that was assigned a value of 100. Positive samples were those ≥ 7.0 AEU, which represented three standard deviations above the lower limit of detection. Kidney tissue was fixed in Bouin's solution for 4 h before being transferred to 70% ethanol and processed into paraffin blocks. Sections were stained with periodic acid-Schiff and hematoxylin. Glomerular hypercellularity was graded on a scale of 0-IV. Grade 0 represented no involvement and grade IV represented severe proliferative glomerulonephritis affecting >90% of glomeruli (Jevnikar et al., 1992Jevnikar A.M. Singer G.G. Brennan D.C. Xu H.W. Kelley V.R. Dexamethasone prevents autoimmune nephritis and reduces renal expression of Ia but not costimulatory signals.Am J Pathol. 1992; 141: 743-751PubMed Google Scholar). To investigate the role of C1q in the clearance of apoptotic keratinocytes in vivo, mice were exposed to varying doses of UVR and the degree of subsequent keratinocyte apoptosis was quantified at different time points. SBC were identified by their distinct appearance on light microscopy Figure 1 and results were expressed as SBC per cm of epidermis. No apoptotic cells were seen in biopsies of normal mouse skin or in skin 24 h after shaving. Initial dose–response experiments performed in wild-type C57BL/6 and C1qa–/– mice demonstrated that maximal keratinocyte apoptosis, without epidermal damage or significant dermal inflammation, was seen 24 h following a UVB radiation dose of 800 mJ per cm2 (data not shown). At this dose, no consistent difference in SBC counts between C1q-deficient and C1q-sufficient controls was demonstrated in four independent experiments Figure 2. To investigate the influence of the genetic background on the role of C1q in apoptotic cell clearance, these experiments were also repeated using C1q-deficient mice on two other genetic strains: 129/Sv and the hybrid strain 129/Sv × C57BL/6. No significant differences were seen between the C1q-deficient and wild-type controls 24 h following a UVB radiation dose of 800 mJ per cm2 Figure 2. In view of the fact that the 800 mJ per cm2 UVB dose produced significant epidermal inflammation 48 h after the exposure rendering the counting of apoptotic cells impossible, the dose radiation was lowered and later time points were studied. Following a UVB dose of 400 or 130 mJ per cm2 no consistent significant difference in SBC counts between the experimental groups was evident up to 96 h following exposure Figure 3a, b. C1q has previously been shown to specifically bind in vitro to apoptotic human keratinocytes (Korb and Ahearn, 1997Korb L.C. Ahearn J.M. C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited.J Immunol. 1997; 158: 4525-4528PubMed Google Scholar). We initially performed immunostaining for murine C1q on apoptotic cultured murine keratinocytes. Twenty-four hours following UVR, significant keratinocyte apoptosis was present in previously confluent keratinocyte cultures Figure 4a, b. Apoptotic but not viable murine keratinocytes stained positively for C1q following UVR exposure in vitro, when incubated for 24 h with 10 µg per ml purified murine C1q Figure 4c, d. To investigate the in vivo significance of this binding, frozen skin sections, taken at various time points following UVR exposure, were examined to determine if murine C1q was present on SBC in vivo. Apoptotic keratinocytes that stained positively for murine C1q were identified in skin sections 24 h after UVR Figure 5a. Notably, these positively stained cells were infrequent in comparison to the degree of keratinocyte apoptosis evident using morphologic criteria on light microscopy. Positively stained cells were not seen in C1q-deficient murine skin sections following UVR Figure 5b. In identical sections, C3 staining on apoptotic keratinocytes was also evident, and demonstrable in both wild-type and C1q-deficient skin sections Figure 6. This finding suggests that the C3 deposition, demonstrated on apoptotic cells following UVR, is a consequence of alternative pathway complement activation.Figure 5Immunostaining for murine C1q on apoptotic murine keratinocytes 24 h after UVR in vivo. (a) C1q positive SBC (arrows) 24 h following a UVB radiation dose of 800 mJ per cm2 in a wild-type murine skin section using immunofluorescence. Positively stained cells were not seen in C1q-deficient murine skin sections following UVR (b). Nonspecific staining of fragments of murine hair shafts is also seen (arrowheads).View Large Image Figure ViewerDownload (PPT)Figure 6Immunostaining for murine C3 on apoptotic murine keratinocytes 24 h after UVR in vivo. C3 positive SBC (arrows) 24 h following a UVB radiation dose of 800 mJ per cm2 in wild-type (a) and C1q-deficient (b) murine skin sections using immunofluorescence. Control staining using a directly labeled isotype control (c).View Large Image Figure ViewerDownload (PPT) C1q-deficient mice on the hybrid genetic background (129/Sv × C57BL/6) developed ANA and glomerulonephritis (Botto et al., 1998Botto M. Dell'Agnola C. Bygrave A.E. et al.Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies.Nat Genet. 1998; 19: 56-59Crossref PubMed Scopus (1200) Google Scholar). In contrast, C1q-deficient C57BL/6 mice do not develop a spontaneous autoimmune phenotype (Botto et al, unpublished observations). It was hypothesized, however, that apoptotic keratinocytes, cleared by an abnormal and potentially pro-immunogenic route in the absence of C1q, could precipitate an autoimmune response. To investigate this hypothesis, cohorts of wild-type and C1q-deficient mice were exposed to chronic UVB radiation. The first cohort of mice comprising 19 C57BL/6 wild-type and 16 C1qa–/– female mice was exposed to a UVB radiation dose of 130 mJ per cm2, three times per week, for a total exposure period of 6 wk. After 1, 2, 3, and 6 wk of exposure, no significant differences in the SBC counts between the experimental groups was evident. Furthermore, neither group developed significant titers of ANA, antidsDNA, antissDNA, or histologic evidence of glomerulonephritis. The second cohort studied comprised 17 C57BL/6 wild-type and 14 C1qa–/– male mice. These mice were exposed to a UVB radiation dose of 400 mJ per cm2, once weekly from the age of 8 wk, for a total period of 7 mo. No significant titers of ANA, antidsDNA, and antissDNA were demonstrable in either group during the exposure period. All mice were sacrificed after the 7 mo exposure period, at the age of 9 mo, and neither group had histologic evidence of glomerulonephritis. In this study we have investigated the clearance of apoptotic murine epidermal keratinocytes or SBC in C1q-deficient mice following UVR exposure. The mechanism by which UVB radiation results in the formation of SBC is not fully understood but is at least partly dependent on p53 (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1302) Google Scholar) and tumor necrosis factor α (TNF-α) (Schwarz et al., 1995Schwarz A. Bhardwaj R. Aragane Y. et al.Ultraviolet-B-induced apoptosis of keratinocytes: evidence for partial involvement of tumor necrosis factor-alpha in the formation of sunburn cells.J Invest Dermatol. 1995; 104: 922-927Crossref PubMed Scopus (245) Google Scholar). Subcutaneous administration of antimurine TNF-α antibody immediately after UVB radiation has been shown to reduce the degree of subsequent SBC formation in Balb/c mice (Schwarz et al., 1995Schwarz A. Bhardwaj R. Aragane Y. et al.Ultraviolet-B-induced apoptosis of keratinocytes: evidence for partial involvement of tumor necrosis factor-alpha in the formation of sunburn cells.J Invest Dermatol. 1995; 104: 922-927Crossref PubMed Scopus (245) Google Scholar). Furthermore, mice deficient in the TNF p55 receptor demonstrate suppressed keratinocyte apoptosis in response to UVB radiation (Zhuang et al., 1999Zhuang L. Wang B. Shinder G.A. Shivji G.M. Mak T.W. Sauder D.N. TNF receptor p55 plays a pivotal role in murine keratinocyte apoptosis induced by ultraviolet B irradiation.J Immunol. 1999; 162: 1440-1447PubMed Google Scholar). Similarly, inactivation of p53 results in reduction of UVB-induced keratinocyte apoptosis in murine skin (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1302) Google Scholar). Although the physiologic fate of SBC is unclear, the most likely hypothesis is that such cells are removed via phagocytosis by neighboring, viable keratinocytes (Young, 1987Young A.R. The sunburn cell.Photodermatol. 1987; 4: 127-134PubMed Google Scholar). Electron microscopy studies of graft versus host disease (De Dobbeleer et al., 1975De Dobbeleer G.D. Ledoux-Corbusier M.H. Achten G.A. Graft versus host reaction. An ultrastructural study.Arch Dermatol. 1975; 111: 1597-1902Crossref PubMed Scopus (41) Google Scholar), Bowen's disease (Olson et al., 1969Olson R.L. Nordquist R.E. Everett M.A. Dyskeratosis in Bowen's disease.Br J Dermatol. 1969; 81: 676-680Crossref PubMed Scopus (18) Google Scholar), and fixed drug eruption (De Dobbeleer and Achten, 1977De Dobbeleer G. Achten G. Fixed drug eruption: ultrastructural study of dyskeratotic cells.Br J Dermatol. 1977; 96: 239-244Crossref PubMed Scopus (16) Google Scholar) have all shown ultrastructural evidence of phagocytosis of dyskeratotic cells by keratinocytes. Passive removal by desquamation is unlikely to play a major physiologic role in eliminating these cells as the majority of SBC are cleared within 72 h and the transit time from the basal layer to the surface of the murine epidermis is approximately 11 d (Potten, 1975Potten C.S. Epidermal transit times.Br J Dermatol. 1975; 93: 649-658Crossref PubMed Scopus (43) Google Scholar). We hypothesized that C1q, deposited on the surface of apoptotic keratinocytes following UVR, was involved in the receptor-mediated clearance of these damaged cells by viable keratinocytes or infiltrating macrophages (Kang et al., 1994Kang K. Hammerberg C. Meunier L. Cooper K.D. CD11b+ macrophages that infiltrate human epidermis after in vivo ultraviolet exposure potently produce IL-10 and represent the major secretory source of epidermal IL-10 protein.J Immunol. 1994; 153: 5256-5264PubMed Google Scholar). Cultured human keratinocytes, rendered apoptotic by either UVB radiation or infection with Sindbis virus, have been previously shown to bind C1q in an antibody-independent manner (Korb and Ahearn, 1997Korb L.C. Ahearn J.M. C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited.J Immunol. 1997; 158: 4525-4528PubMed Google Scholar). Our findings demonstrate that, following UVR exposure, apoptotic murine keratinocytes also bind murine C1q in vitro. In addition, we show for the first time the presence of C1q on apoptotic keratinocytes in the murine epidermis following UVR exposure in vivo. Using our acute UVR exposure model, however, no difference in the rate of clearance of SBC was found in C1q-deficient mice from three different genetic backgrounds, compared to strain-matched wild-type controls. This finding suggests that murine C1q does not play a critical role in the physiologic clearance of apoptotic keratinocytes in vivo. In addition to C1q, we were also able to demonstrate C3 deposition on apoptotic keratinocytes following UVR exposure. Cultured human epidermal keratinocytes have previously been shown to synthesize C3 in vitro (Basset-Seguin et al., 1990Basset-Seguin N. Caughman S.W. Yancey K.B. A-431 cells and human keratinocytes synthesize and secrete the third component of complement.J Invest Dermatol. 1990; 95: 621-625Abstract Full Text PDF PubMed Google Scholar), and C3 production is augmented in the presence of the pro-inflammatory cytokines γ-interferon (γ-IFN) and TNF-α (Terui et al., 1997Terui T. Ishii K. Ozawa M. Tabata N. Kato T. Tagami H. C3 production of cultured human epidermal keratinocytes is enhanced by IFNgamma and TNFalpha through different pathways.J Inves
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