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Department of Pediatrics and Adolescent Medicine (P.F.-P., G.S., K.-M.D., M.W.), University of Ulm, D-89075 Ulm, Germany; University Children’s Hospital (H.H.), University of Würzburg, D-97080 Würzburg, Germany; Clinic for Plastic Surgery (A.K.H.), D-89073 Ulm, Germany; and Department of Pathology (P.M.), University of Ulm, D-89081 Ulm, Germany
Address all correspondence and requests for reprints to: Prof. Dr. Martin Wabitsch, Pediatric Endocrinology, Department of Pediatrics and Adolescent Medicine, University of Ulm, Prittwitzstrasse 43, 89075 Ulm, Germany. E-mail: martin.wabitsch{at}medizin.uni-ulm.de.
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Abstract |
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Objective: We have analyzed sc adipose tissue from a 5-yr-old girl with ongoing fat loss by immunohistochemistry. Using cultured human preadipocytes and adipocytes, we elucidated a possible mechanism leading to adipocyte loss in this patient.
Results: Analysis of adipose tissue samples of the patient with acquired lipodystrophy obtained from skin areas affected by panniculitis suggested that loss of adipocytes was mediated by CD95-induced apoptosis. Regression of adipose tissue was accompanied by lymphohistiocytic infiltration/inflammation and increased serum levels of inflammatory cytokines interferon-
and TNF-
. In vitro studies with human adipocytes demonstrated that interferon- and TNF- are able to up-regulate CD95 expression and enhance CD95-death-inducing signaling complex formation resulting in a robust sensitization for CD95-mediated apoptosis.
Conclusion: We have identified here a possible mechanism responsible for the loss of adipocytes by apoptosis in autoimmune lipodystrophy.
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Introduction |
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In both acquired generalized and partial lipodystrophy, the loss of adipose tissue typically occurs during childhood and adolescence. In the generalized form, loss of fat affects large areas of the body, particularly the face, arms, and legs (1). About 25% of affected patients have associated autoimmune disorders. In another 25%, the onset of AGL is heralded by an episode of lobular panniculitis (2). Initially, these lesions heal, with localized loss of sc adipose tissue, but subsequently fat is lost from almost all sc regions, eventually causing generalized lipodystrophy (2). In APL, fat loss affects the face, neck, arms, thorax, and upper abdomen. Some patients develop autoimmune diseases after the onset of APL (1).
The pathogenic basis of acquired lipodystrophies and the mechanism of fat loss are still poorly understood. The association with autoimmune diseases in some forms of AGL or APL suggests that the pathogenic mechanism may be an expression of autoimmunity (1, 3, 4, 5, 6). Whether autoimmunity is triggered by viral or other infections is still not clear (1).
Hashimoto’s thyroiditis and type 1 diabetes are two examples of autoimmune diseases in which apoptosis of endocrine cells seems to occur via CD95 receptor activation (7, 8). Apoptosis of adipocytes might be one possible mechanism of fat loss in AGL. Our group has recently demonstrated receptor-mediated apoptosis of human adipocytes and preadipocytes (9). In the present report, we show that the CD95 system is involved in sc adipose tissue loss in a patient with an acquired lipodystrophy. Using a human preadipocyte cell line as a model, we could further elucidate the mechanism leading to activation of the CD95 pathway under pathogenic conditions.
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Patients and Methods |
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Cell culture medium, fetal bovine serum, and antibiotics were obtained from Life Technologies, Inc. (Karlsruhe, Germany). TNF- was purchased from PeproTech (London, UK), interferon- (IFN-) was from Roche (Mannheim, Germany). Rosiglitazone (BRL 49653) was a kind gift from Smith-Kline-Beecham (London, UK). Recombinant human insulin was kindly provided by Novo Nordisk (Gentofte, Denmark). Other chemicals and reagents were purchased from Sigma Chemical Co. (Taufkirchen, Germany).
Case report
A 5-yr-old girl presented with a 4-month history of a rapidly progressing acquired lipodystrophy. The patient described short-lasting localized pain on the skin followed by the development of a dent resulting from a regional loss of sc tissue. This sequence of events recurred many times at different skin areas. Because the patient was obese (weight, 28.7 kg; height, 115 cm) with an increased amount of sc adipose tissue, these skin alterations were easily detectable. At presentation in our hospital, the patient had an almost complete lack of sc adipose tissue at the lower legs and buttocks with superficial veins and muscles appearing prominent. Ultrasound examination revealed an almost complete loss of sc adipose tissue at the affected sites. Multiple dents of various sizes with a lack of sc adipose tissue (as shown by ultrasound examination) were found at the upper legs, the abdomen, the thorax, and the arms. Lipodystrophy was not yet generalized, and the face seemed not to be affected by the disease so far. According to ultrasound examination, intraabdominal and retroperitoneal adipose tissue sites seemed not to be affected by regional adipose tissue losses. Triglyceride concentrations in fasted serum of the patient varied between 4.2 and 18.7 mmol/liter. Fasting serum level of insulin was 0.88 nmol/liter (normal range, <0.1 nmol/liter) indicating severe fasting hyperinsulinemia. No signs of acanthosis nigricans were present. Fasting glucose plasma levels were less than 5.6 mmol/liter, and an oral glucose tolerance test still showed normal glucose tolerance. Serum levels of liver enzymes alanine aminotransferase and aspartate aminotransferase were normal, whereas ultrasound examination of the liver showed hepatomegaly with mild signs of liver steatosis. So far the patient had no clinical or serological evidence of another autoimmune disease or infectious disease. White blood cells counts and serum C-reactive protein levels were normal.
Several skin biopsies of 2 x 2 mm2 were taken from different affected and unaffected skin areas by stamp. Informed consent was obtained from the mother of the patient. The study was approved by the ethical committee of the University of Ulm.
Histology and immunohistochemistry
Histopathology was analyzed on formalin-fixed, paraffin-embedded tissue. Routine paraffin sections of 0.5 µm were stained with routine stains, e.g. hematoxilin/eosin, Giemsa, periodic acid-Schiff.
For immunohistochemistry, serial, 2-µm-thick cryosections of snap-frozen, unfixed biopsy material were immediately fixed in ice-cold acetone for 10 min, air dried, and incubated for 1 h with the following mouse antihuman monoclonal antibodies in appropriate dilutions: CD3(Leu4) IgG1 isotype (Becton Dickinson, Mountain View, CA), CD4(Okt4) IgG2a isotype, CD8(C8/144B) IgG1 isotype (Dako, Copenhagen, Denmark), CD11c(BU15) IgG1 isotype (Immunotech, Marseille, France), CD57(NK-1) IgG1 isotype (Dako), CD95(anti-APO-1) IgG1 isotype (Dako), and CD95 ligand (CD95L) clone G 247-4 IgG1 isotype (PharMingen, San Diego, CA). Anti-HLA-A, -B, -C clone W6/32 IgG1 isotype and anti-HLA-DR -chain clone TAL.1B5 IgG1 isotype was generously contributed by Gerhard Moldenhauer (Deutsches Krebsforschungszentrum, Heidelberg, Germany). A purified rabbit antiserum to bovine S100p reactive with human S100p (Dako) was also used. Bound primary antibody was detected via goat antimouse or antirabbit Ig conjugated to peroxidase-labeled dextran polymer in Tris-HCl buffer containing carrier protein (EnVision; Dako). 3-Amino-9-ethylcarbazole (Sigma) was used for substrate. Counterstain was performed with hemalum. Controls were performed by omitting the first-step antibody and yielded negative results.
Measurement of cytokines in human serum samples
Circulating IFN-, TNF-, and soluble CD95L (sCD95L) were measured by a commercial ELISA (from Biozol, Eching, Germany, for IFN- and TNF-; from MBL Co. Ltd., Nagoya, Japa, for sCD95L). The sensitivity of the ELISA was 1.5 pg/ml for IFN-, 3 pg/ml for TNF-, and 50 pg/ml for sCD95L. Normal values in healthy subjects are below these detection limits as indicated by the manufacturers.
Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) staining
Apoptosis events were detected by TUNEL as previously described (10).
Cell culture
SGBS preadipocytes (11) were cultured in DMEM/Ham’s F12 (1:1) containing 33 µM biotin, 17 µM pantothenate, antibiotics (serum-free, basal medium), and 10% fetal bovine serum. Adipogenic differentiation was induced after reaching near confluence. Cells were washed three times with PBS and cultured in serum-free, basal medium supplemented with 10 µg/ml iron-poor transferrin, 10 nM insulin, 200 pM T3, and 1 µM cortisol. For the first 4 d, 2 µM rosiglitazone (BRL 49653), 250 µM isobutylmethylxanthine, and 25 nM dexamethasone was added. The medium was changed every 4 d. Morphologically differentiated adipocytes were obtained after 14 d. The number of differentiated cells was estimated in the monolayers by direct counting using a net micrometer, and cultures were used for experiments when differentiation rate was at least 85%.
Induction and analysis of apoptosis
After washing cells three times with PBS, apoptosis was induced in serum-free, basal medium by adding 1 µg/ml of an agonistic CD95 monoclonal antibody (anti-APO-1, IgG3) (12), 10–8 M TNF-, and 1000 U/ml IFN-.
Quantitative determination of apoptosis was performed as described elsewhere (9, 13).
4',6-Diamidino-2-phenylindole (DAPI)/Nile Red staining
SGBS adipocytes grown on chamber slides (Becton Dickinson, Heidelberg, Germany) were used for staining nuclei and lipid droplets to show morphological signs of apoptosis. After induction of apoptosis for 24 h, staining was performed as described previously (9).
Detection of CD95 expression
SGBS adipocytes grown in 12-well plates were used for determination of CD95 expression. Cells were treated for 24 or 48 h with 10–8 M TNF- and 1000 U/ml IFN-. After washing twice with PBS, monolayers were incubated in the dark for 30 min at 4 C with polyethylene (PE)-conjugated CD95 clone DX2 isotype IgG1 (Dako) or IgG1 control antibody (Dako). After washing once with PBS, cells were carefully detached using Accutase and directly analyzed by flow cytometry. Mean fluorescence intensity (MFI) ratio was calculated according to the formula: MFI (CD95-PE)/MFI (isotype-PE). Comparisons between medium control and different treatments were made using Student’s t test.
Western blotting and death-inducing signaling complex (DISC) analysis
Adipocytes grown in 75-cm2 flasks were stimulated with 10–8 M TNF- and 1000 U/ml IFN- for 6, 24, and 48 h. Cells were lysed for 15 min at 4 C in lysis buffer (30 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 1 mM phenylmethylsulfonyl fluoride; and 1 µM dithiothreitol) followed by high-speed centrifugation, and 50 µg of lysate was separated on a 10–20% gradient SDS-PAGE and electroblotted onto Hybond ECL nitrocellulose membrane (Amersham, Braunschweig, Germany). Membranes were blocked for 1 h in PBS supplemented with 5% milk powder and 0.1% Tween 20. Membranes were stained overnight at 4 C with the first antibody, followed by 1-h incubation with the horseradish peroxidase-conjugated second antibody, and detection was done by enhanced chemiluminescence (Amersham). The following antibodies were used: CD95 (C20) rabbit IgG (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), caspase-8 (clone 12F5) isotype IgG2b (Alexis, Grunberg, Germany), Fas-associated death domain (FADD) (clone 1) isotype IgG1 (BD Transduction Laboratories, Heidelberg, Germany), and -tubulin (DM1A) isotype IgG1 (Oncogene, Bad Soden, Germany). Horseradish peroxidase-conjugated goat antimouse IgG and goat antirabbit IgG were obtained from Santa Cruz.
For DISC analysis, SGBS adipocytes were incubated with 10–8 M TNF- and 1000 U/ml IFN- for 48 h, and 5 x 105 adipocytes were treated with cross-linking CD95 antibody (anti-APO-1 antibody, IgG3, 1 µg/ml) for 10 min at 37 C. Control cells were incubated in the absence of CD95 antibody. Cells were washed once with ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 1 mM phenylmethylsulfonyl fluoride; and 1 µM dithiothreitol), followed by high-speed centrifugation for 10 min at 4C. Anti-APO-1 (1 µg/ml) was added to the control cells. To precipitate CD95, 10 µl of pan mouse IgG Dynabeads (Dynal Biotech, Hamburg, Germany) were added and incubated for 4 h at 4 C. Beads were then washed three times with 1 ml washing buffer (1% IGEPAL CA-630, 500 mM NaCl, and 50 mM Tris-HCl, pH 8) and once with 25 mM Tris-HCl (pH 7.5). Beads were resuspended in 6x SDS-reducing sample buffer, boiled for 5 min at 95 C, and separated by Dynal magnetic particle concentrators. The supernatant was separated by 10–20% gradient SDS-PAGE, followed by Western blotting as described above.
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Results |
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). In the newly affected gluteal region (Fig. 1B), loss of adipose tissue is heralded by lobular panniculitis. At the time of the patient’s presentation, serum levels of inflammatory cytokines IFN- and TNF- as well as soluble CD95L were increased (IFN-, 3 pg/ml; TNF-, 632 pg/ml; and sCD95L, 150 pg/ml).
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, C–E); CD57+ natural killer cells (Fig. 1F); and CD11c+ macrophages (Fig. 1G). Many macrophages contained large lipid droplets suggesting phagocytosis of adipocytes (Fig. 1G). Molecule expression of HLA-A, -B, and -C (Fig. 1I) and HLA-DR (Fig. 1J) was present in adipocytes in infiltrated areas. In addition, immunostaining for S100 protein (Fig. 1H) reveals dendritic interstitial cells being part of the inflammatory infiltrate and adipocytes. Both cell types constitutively express S100 protein. Taken together, these results demonstrate that loss of adipose tissue is accompanied by lymphohistiocytic infiltration/inflammation.
We hypothesized that loss of adipocytes in acquired lipodystrophy may be mediated by programmed cell death. TUNEL staining of sc adipose tissue samples taken from the gluteal region with ongoing fat loss revealed TUNEL-positive events (Fig. 2A) demonstrating apoptosis of adipocytes in this patient with acquired lipodystrophy. No TUNEL-positive cells were detected in sc adipose tissue samples from unaffected body regions from the patient or in samples from healthy patients (data not shown). There are several known ways of apoptosis induction. One main mechanism involves activation of death receptors by their matching death ligands. The CD95 system has been described to play an important role in the pathogenesis of other autoimmune diseases (7, 14, 15). By immunohistochemical analysis we showed that CD95 as well as CD95L are expressed in sc adipose tissue from body regions with ongoing fat loss (Fig. 2, B and C). Although CD95 expression could be localized to lipid-filled adipocytes (Fig. 2B), expression of CD95L was restricted to cells of the inflammatory infiltrate (Fig. 2C). Adipose tissue sections from unaffected body regions from the patient with acquired lipodystrophy as well as samples from healthy patients stained negative for both CD95 and CD95L (data not shown).
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and TNF- were increased in our patient, we have studied whether both of these cytokines could lead to a sensitization for CD95-triggered apoptosis in vitro. SGBS preadipocytes and adipocytes were treated with cross-linking CD95 antibody (anti-APO-1) and subsequently evaluated for apoptosis induction by flow cytometry measuring DNA fragmentation (Fig. 3A). As shown earlier, anti-APO-1 alone induced low rates of specific apoptosis in both cell types (
5% after 72 h) (9). In preadipocytes, IFN- or TNF- alone did not induce apoptosis, and a combination of both cytokines resulted only in very low amounts of specific apoptosis (<7% after 72 h). Costimulation with anti-APO-1 induced approximately 13% specific apoptosis. In SGBS adipocytes, however, stimulation with IFN- and TNF- resulted in a robust sensitization for CD95-mediated apoptosis (at 72 h: IFN-, 5%, TNF-, 5%; IFN- plus TNF-, 15%; and IFN- plus TNF- plus anti-APO-1, 30%). To further demonstrate that cell death occurs via apoptosis, cells were treated for 24 h with either serum-free, basal medium alone or with anti-APO-1, IFN-, and TNF-. A combined staining with DAPI and Nile Red revealed normal nuclear morphology and lipid vacuoles after incubation in medium alone, whereas nuclear condensation and loss of incorporated lipids appeared only after cytokine treatment and CD95 triggering (Fig. 3B).
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and TNF- and subsequently evaluated for CD95 expression by flow cytometry (Fig. 4). CD95 is constitutively low-level expressed on adipocytes in vitro (Fig. 4A). After 48 h, incubation with IFN- or TNF- resulted in a clear up-regulation of CD95 expression by approximately 2.5-fold (Fig. 4B). An approximately 4-fold increase in CD95 expression was detected after combined treatment with both cytokines (Fig. 4B). Longer periods of treatment did not further increase CD95 expression (data not shown).
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and TNF- resulted in an up-regulation of CD95 expression and a sensitization for CD95-mediated apoptosis in this murine cell line (data not shown).
To assess whether up-regulation of CD95 expression influences transduction of the apoptotic signal, we next analyzed formation of the CD95 DISC including CD95, caspase-8, and FADD (Fig. 5A). After pretreatment with IFN- and TNF- for 48 h, SGBS adipocytes were incubated with a cross-linking agonistic CD95 antibody (anti-APO-1) or left untreated. After cell lysis, CD95 was precipitated, and subsequently Western blot analysis was performed to determine compounds bound to the receptor. In the absence of CD95 cross-linking, no association between caspase-8 and the CD95 receptor was detected. However, low amounts of FADD were associated with CD95 in the absence of CD95 cross-linking. After CD95 triggering, caspase-8 and FADD were recruited to the DISC. Recruitment of caspase-8 and FADD to the CD95 receptor was strongly increased after treatment with IFN- and TNF-. Increasing amounts of caspase-8 and FADD at the DISC correspond to increased expression of CD95 after IFN- and TNF- treatment as shown by restaining the membranes with a CD95 antibody. Equal loading of protein was controlled by performing Western blot analysis on protein lysates after immunoprecipitation using an anti--tubulin antibody (data not shown). To show that increased CD95 expression is the only factor leading to increased DISC formation, we have treated SGBS adipocytes with IFN- and TNF- and tested for caspase-8 and FADD expression (Fig. 5B). Expression of both proteins remained unchanged during cytokine treatment.
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Discussion |
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In our patient, loss of fat tissue was associated with lobular panniculitis. Twenty-five percent of cases with AGL are preceded by local panniculitis (1, 2). Immunohistochemical investigation of adipose tissue sections taken from affected body regions revealed inflammatory infiltrates including CD3+, CD4+, CD8+ T cells, CD57+ natural killer cells, and CD11c+ macrophages. Strong molecule expression of HLA-A, -B, and -C and HLA-DR in adipocytes underlines ongoing immunological processes during fat loss. However, the initial cause of HLA-DR up-regulation and the presented antigen were not identified. Most of the macrophages within one section were filled with lipids, suggesting phagocytosis of adipocytes. Charriere et al. (23) have shown recently that preadipocyte and macrophage phenotypes are very similar and that preadipocytes have the potential to be very efficiently and rapidly converted into macrophages. Because of these new findings, adipocytes in the patient could be incorporated by macrophages of the hematopoietic lineage or from preadipocytes transdifferentiated into macrophages.
Loss of fat in our patient with acquired lipodystrophy was associated with the expression of CD95 and CD95L in affected sc adipose tissue. CD95 was localized to adipocytes, whereas CD95L expression was restricted to the inflammatory infiltrate. Screening normal human tissues, Leithäuser et al. (24) have shown rare but detectable amounts of CD95 in connective tissue but complete absence of CD95 in mature, lipid-filled adipocytes in adipose tissue from healthy patients. This suggests that expression of CD95 is induced in adipose tissue of the patient with acquired lipodystrophy probably by cytokines produced by the inflammatory infiltrate. Increased serum levels of IFN- and TNF- were measured in the patient; both cytokines are not detectable in serum samples of healthy individuals. Very high amounts of TNF- (632 pg/ml) were detected, whereas IFN- was slightly increased (3 pg/ml). However, one would expect even higher levels of both cytokines locally, in affected adipose tissue areas. Cytotoxic cytokines such as IFN- and TNF- are known to induce or to up-regulate CD95 expression in some cell lines and to thereby sensitize these cells for CD95-mediated apoptosis (16, 25, 26, 27, 28, 29, 30). In our patient, adipocytes might be sensitized for CD95-mediated apoptosis after up-regulation of receptor expression. Fat cells finally undergo apoptosis after binding of CD95L, which is produced by infiltrating immune cells.
To further investigate whether up-regulation of CD95 by IFN- and TNF- is a common mechanism sensitizing human fat cells for apoptosis, we have used human SGBS preadipocytes and adipocytes (11).
In our in vitro studies, treatment with IFN- and TNF- clearly sensitized SGBS adipocytes for CD95-triggered apoptosis as shown by typical biochemical and morphological signs of apoptosis. As one possible mechanism of sensitization, we have identified up-regulation of CD95 expression.
IFN- and TNF- are known to sensitize other cell types for CD95-mediated apoptosis including cancer cell lines (16, 25, 26, 27), a salivary ductal cell line (28), the seminiferous epithelium (29), and also pancreatic ß-cells (30). In these cells, sensitization for apoptosis was associated with CD95 up-regulation. In a colon cancer cell line, enhancement of CD95 expression was caused by activation of nuclear factor-
B after IFN- and TNF- treatment (27). Kim et al. (31) have reported potentiation of CD95-mediated apoptosis after IFN- in lung epithelial cells by increasing caspase-8 expression via an IFN- response element. In our experiments, caspase-8 expression in adipocytes remained constant upon cytokine treatment.
Finally, we could demonstrate here that increased expression of CD95 after IFN- and TNF- treatment was associated with increased DISC formation as shown by coimmunoprecipitation assays. Changes in CD95 sensitivity are often associated with changes in formation of the CD95-DISC. For example, development of CD95 resistance in activated, antigen-specific T cells upon repeated stimulation with antigen is associated with impairment of DISC formation (32). In a carcinoma cell line, protection from CD95-induced apoptosis by hepatocyte growth factor was mediated by suppression of DISC formation (33). Vice versa, improvement of DISC formation by a novel protein, Insulinoma-Glucagonoma clone 20 (IG20), enhances death receptor-induced apoptosis (34).
In conclusion, our findings suggest that loss of adipose tissue in the presented patient with autoimmune lipodystrophy is mediated by the CD95 system. Showing that inflammatory cytokines (IFN- and TNF-) up-regulated CD95 expression and enhanced CD95-DISC formation in vitro, we have identified one possible mechanism that could lead to loss of adipocytes by apoptosis in autoimmune lipodystrophy.
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Footnotes |
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Abbreviations: AGL, Acquired generalized lipodystrophy; APL, acquired partial lipodystrophy; CD95L, CD95 ligand; DAPI, 4',6-diamidino-2-phenylindole; DISC, death-inducing signaling complex; FADD, Fas-associated death domain; IFN-, interferon-; MFI, mean fluorescence intensity; PE, polyethylene; sCD95L, soluble CD95L; TUNEL, terminal deoxynucleotidyltransferase dUTP nick end labeling.
This work was supported by the German Research Association (Deutsche Forschungsgemeinschaft) (WA1096/3-2).
Received April 5, 2005.
Accepted December 13, 2005.
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References |
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in human neuroblastoma cell lines through the CD95/CD95L autocrine circuit. Cell Death Differ 6:652–660[CrossRef][Medline] upregulates apoptosis-related molecules and enhances Fas-mediated apoptosis in human cholangiocarcinoma. Int J Cancer 100:445–451[CrossRef][Medline] on the susceptibility to Fas (CD95/APO-1)-mediated cell death in human hepatoma cells. Cancer Immunol Immunother 50:23–30[CrossRef][Medline] accelerates NF-B-mediated apoptosis through enhancement of Fas expression in colon cancer cells. Cell Death Differ 10:718–728[CrossRef][Medline] and tumor necrosis factor induce Fas expression and anti-Fas mediated apoptosis in a salivary ductal cell line. Clin Exp Rheumatol 18:311–318[Medline] and IFN- regulate expression and function of the Fas system in the seminiferous epithelium. J Immunol 165:743–749 and TNF- increase vulnerability of pancreatic ß cells to autoimmune destruction. J Autoimmun 20:303–312[CrossRef][Medline] in A549 lung epithelial cells: enhancement of caspase-8 expression through IFN-response element. Cytokine 20:283–288[CrossRef][Medline]Read all eLetters
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