ARTICLE |
Correspondence to: Giorgio Stassi, Dept. of Surgical, Anatomical and Oncological Sciences, Human Anatomy Section, U. of Palermo, Via del Vespro 129, 90127 Palermo, Italy.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Farber's disease (FD) is a rare genetic disorder caused by ceramidase deficiency, which results in ceramide accumulation in lung, liver, colon, skeletal muscle, cartilage, and bone. Although this disease has been symptomatically characterized, little is known about its molecular pathogenetic process. Because recent studies reported that ceramide accumulation induces GD3 ganglioside formation and apoptosis, we investigated, in tissue obtained via colonoscopy from seriously involved patients, the possible involvement of ceramide in FD colonocyte destruction. Histochemical and TUNEL analyses of paraffin-embedded sections revealed that 45 ± 4.3% of FD colonocytes showed morphological signs of apoptosis compared with the 8 ± 2.3% of constitutive epithelial cell death. Importantly, immunohistochemical study for pro-apoptotic factors showed that GD3 accumulation co-localized with active caspase-3 and cleaved K18 in FD colon tissue. These findings provide evidence for a role of the apoptotic ceramide pathway in the pathogenesis of FD. (J Histochem Cytochem 48:5762, 2000)
Key Words: apoptosis, caspases, active caspase-3, GD3 ganglioside, K18, Farber disease
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FARBER DISEASE (FD) is a rare lysosomal storage disorder, also called Farber lipogranulomatosis, characterized by an inherited deficiency of acid ceramidase activity (
Although joint pain is the most common symptom, more severely involved patients may have respiratory, gastrointestinal, and neurological impairments (
Ceramide accumulation results from impaired ceramide hydrolysis due to loss of function mutations of the acid ceramidase gene (
Ceramide is a cell signaling molecule involved in a variety of biological effects in different cell types, including cell proliferation and differentiation, inflammation, and apoptosis (
Caspase-3 is one of the most specific proteases involved in the apoptosis mechanism (
In this study we investigated whether ceramide accumulation in FD triggers the apoptotic machinery in colon epithelial cells, thus contributing to the pathogenesis of this genetic disorder.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Specimens
Colon mucosal biopsies were obtained at the time of colonoscopy. The diagnosis of FD was based on clinical criteria and was confirmed by acid ceramidase deficiency in cultured white blood cells (
Histochemistry
Tissues from each subject were fixed and stained with hematoxylin and eosin (H&E) and periodic acidSchiff (PAS; SigmaAldrich, St Louis, MO) (
Immunohistochemistry
Immunohistochemical stains were performed on paraffin-embedded tissue sections (7 µm) obtained from FD colon and normal human colon specimens. Sections were dewaxed in xylene and dehydrated through graded alcohols (100% to 30%). Endogenous peroxidase was inhibited by exposing sections to 3% H2O2 for 10 min at RT. Then sections were rinsed for 10 min in distilled water. Before starting the immunostaining procedure, sections were treated with citric acid (2 g/liter, pH 6.0) for 15 min in the microwave for antigen retrieval.
Sections were incubated for 10 min with TBS containing 1% BSA to block aspecific staining. After elimination of excess serum, sections were exposed for 1 h to specific antibodies anti-Fas (DX2, mouse IgG1; PharMingen, San Diego, CA), anti-GD3 (S2-566, mouse IgM; Seikagaku, Tokyo, Japan), anti-CPP32 (clone 19, mouse IgG2a; Transduction Laboratories, Lexington, KY), anti-caspase-3, active form (polyclonal rabbit; kindly provided by Merk Frosst, Centre for Therapeutic Research, Merck Research Laboratories, Kirkland, Quebec, Canada), anti-M30 cytodeath (mouse IgG2b; Boehringer Mannheim, Mannheim, Germany) (
In Situ Apoptosis Detection
TdT-mediated dUTP nick end-labeling (TUNEL) reaction was detected in serial paraffin-embedded sections. After dewaxing, sections were washed twice with PBS and cells permeabilized with 0.1% Triton X-100, 0.1% sodium citrate for 2 min on ice. Then, in situ apoptosis detection was performed with an In Situ Cell Death Detection AP Kit (Boehringer Mannheim; Indianapolis, IN). DNA strand break detection was done with an antifluorescein antibody conjugated with alkaline phosphatase (AP) and revealed by 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Dakopatts, Glostrup, Denmark). Eosin was used as counterstain. A positive control was carried out by pretreating the specimens with DNase I (1 µg/ml) to introduce nonspecific strand breaks (not shown). By contrast, the negative control was subjected to the same staining for TUNEL without terminal deoxynucleotidyl transferase (TdT) (
Quantification of Apoptotic Events
H&E-, PAS-, and TUNEL-stained sections were examined for the scoring of cells undergoing apoptosis, detected morphologically as membrane-bound apoptotic bodies and enzymatically as dark blue staining. The number of apoptotic events was randomly counted in 40 high-power microscopic fields (x40). Approximately 500 nuclei per colon mucosal biopsy were counted. The number of apoptotic cells was expressed as number per 100 nuclei. Comparison between the two groups was performed by Student's t-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ceramide Accumulation
H&E staining showed normal crypt architecture in both normal and FD colon tissues, whereas lymphocytes, plasma cells, and eosinophils were numerous in the lamina propria of FD colon mucosa compared to colon of healthy individuals (Figure 1a and Figure 1b).
|
In FD tissue, PAS staining showed an abundant accumulation of polysaccharides, lipids, and glycosphingolipids containing ceramide precursors that are represented by strong purple-red staining (Figure 1d).
Ceramide Accumulation Induces Abundant Morphological Changes Associated with Apoptosis in FD Colonocytes
H&E and PAS scoring analyses showed a large number of late-stage apoptotic cells in FD colon (Figure 1b and Figure 1d, arrowheads). In situ TUNEL detection revealed massive early- and late-stage DNA fragmentation stained in dark blue (Figure 1h, arrowheads) compared to a few apoptotic epithelial cells found in normal colon (Figure 1g). The mitotic activity of stem cells at the base of the crypts produces a continuous supply of new cells, which progress to the mucosal surface where they mature before degeneration by apoptosis.
Most of the normal colon epithelial cells showing morphological evidence of apoptosis (8 ± 2.3% of the total) were localized to the mucosal surface (Figure 2). In contrast, whereas only 0.8 ± 0.2% of crypt cells were apoptotic in normal colon, a massive number of colonocytes (45 ± 4.3%) were apoptotic in crypts from FD colon (p<0.001) (Figure 1g and Figure 1h, arrowheads, and Figure 2). These findings suggest that ceramide accumulation and triggering of apoptosis machinery may play an important role in FD cell destruction.
|
Active Caspase-3 Cleaves K18, Leading to Cell Death In Vivo
We then investigated whether ceramide-induced apoptosis in FD colonocytes showed involvement of caspase activation. Interestingly, analyses of serial colon sections shows that the active form of caspase-3 in colonocytes was mostly evident in colon crypt areas containing abundant reactivity for M30 (Figure 3bd and Figure 4c, Figure d), an antibody that specifically recognizes a caspase cleavage site in K18 that is not detectable in native K18 of normal cells (
|
GD3 Gangloside Co-localizes with Active Caspase-3 and Cleaved K18
Accumulation of GD3 ganglioside is an early event in Fas- and ceramide-induced apoptosis, responsible for apoptotic signal generation (
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although FD is genetically characterized, the pathogenic mechanisms determining cell tissue destruction are still not clear. Ceramide accumulation in FD cells has been classically regarded as the major pathogenic factor correlated with the severity of the disease (
Apoptosis has been proposed as a key pathogenic mechanism in several genetic, degenerative, and autoimmune diseases (
The interaction of Fas and its ligand is a major apoptosis-inducing mechanism. Fas is a surface receptor expressed on many different cell types (
Fas is rarely expressed in FD colonocytes, suggesting that is not involved in colon epithelial cell apoptosis. In contrast, GD3 ganglioside is abundantly accumulated, probably due to the deficient acid ceramidase activity. It is likely that in these cells GD3 acts as an inducer of caspase activation, promoting cleavage of a number of substrates that eventually results in cell death.
Recent studies support the idea that caspase-3 activation is responsible for fragmentation of keratins in apoptotic epithelial cells by specific cleavage (
In summary, GD3 synthesis induced by a high concentration of ceramide may alter mitochondrial function and promote cell destruction in a caspase-dependent manner, explaining the pathogenic mechanism of cell injury in FD colonocytes. Apoptosis along the entire colon epithelium down into the crypts may be a key pathogenetic factor in the persistent diarrhea and severe gastrointestinal lesions that occasionally occur in FD (
![]() |
Acknowledgments |
---|
Supported by Telethon-Italy Grant E.735 (to GS) and MURST 60% (to FF and GZ).
We are grateful to Drs S. Accomando (Pediatric Institute, University of Palermo), V. Marcianò, and L. Licata (Human Anatomy Section, University of Palermo) and G. Balsano (Institute of General Surgery, University of Palermo) for providing surgical specimens and helpful discussions and advice.
Received for publication May 27, 1999; accepted August 12, 1999.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abenoza P, Sibley RK (1987) Farber's disease: a fine structural study. Ultrastruct Pathol 11:397-403[Medline]
Anjum R, Ali AM, Begum Z, Vanaja J, Khar A (1998) Selective involvement of caspase-3 in ceramide induced apoptosis in AK-5 tumor cells. FEBS Lett 439:81-84[Medline]
Ballou LR, Laulederkind SJF, Rosloniec EF, Raghow R (1996) Ceramide signalling and the immune response. Biochim Biophys Acta 1301:273-278[Medline]
Burck U, Moser HW, Goebel HH, Grutter R, Held KR (1985) A case of lipogranulomatosis Farber: some clinical and ultrastructural aspects. Eur J Pediatr 143:203-208[Medline]
Caulìn C, Salvesen GS, Oshima R (1997) Caspase cleavage of keratin 18 and reorganization of intermediate filament during epithelial cell apoptosis. J Cell Biol 138:1379-1394
Chatelut M, Feunteun J, Harzer K, Fensom AH, Basile JP, Salvayre R, Levade T (1996) A simple method for screening for Farber disease on cultured skin fibroblasts. Clin Chim Acta 245:61-71[Medline]
Chen WW, Decker GL (1982) Abnormalities of lysosomes in human diploid fibroblasts from patients with Farber's disease. Biochim Biophys Acta 718:185-192[Medline]
Cifone MG, Roncaioli P, De Maria R, Camarda G, Santoni A, Ruberti G, Testi R (1995) Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal. EMBO J 14:5859-5868[Abstract]
De Maria R, Lenti L, Malisan F, D'Agostino F, Tomassini B, Zeuner A, Rippo MR, Testi R (1997) Requirement for GD3 ganglioside in CD95- and ceramide-induced apoptosis. Science 277:1652-1655
De Maria R, Rippo MR, Schuchman ES, Testi R (1998) Acidic sphingomyelinase is necessary for Fas-induced GD3 ganglioside accumulation and efficient apoptosis of lymphoid cells. J Exp Med 187:897-902
Fujiwaki T, Hamanaka S, Koga M, Ishihara T, Nishikomori R, Kinoshita E, Furusho K (1992) A case of Farber disease. Acta Paediatr Jpn 34:72-79[Medline]
Genestier L, Prigent AF, Paillot R, Quemeneur L, Durand I, Banchereau J, Revillard JP, Bonnefoy Bérard N (1998) Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis. J Biol Chem 273:5060-5066
Giordano C, Stassi G, De Maria R, Todaro M, Richiusa P, Papoff G, Ruberti G, Bagnasco M, Testi R, Galluzzo A (1997) Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto's thyroiditis. Science 275:960-963
Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309-1312
Haraoka G, Muraoka M, Yoshioka N, Wakami S, Hayashi I (1997) First case of surgical treatment of Farber's disease. Ann Plast Surg 39:405-410[Medline]
Koch J, Gartner S, Li C, Quintern LE, Bernardo K, Levran O, Schnabel D, Desnick RJ, Schuchman E, Sandhoff K (1996) Molecular cloning and characterization of a full-length complementary DNA encoding human acid ceramidase. J Biol Chem 271:33110-33115
Koga M, Ishihara T, Uchino F, Fujiwaki T (1992) An autopsy case of Farber's lipogranulomatosis in a Japanese boy with gastrointestinal involvement. Acta Pathol Jpn 42:42-48[Medline]
Leers MPG, Kölgen W, Björklund B, Bergman T, Tribbick G, Persson B, Björklund P, Ramaekers FCS, Björklund B, Nap M, Jörnval H, Schutte B (1999) Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 187:567-572[Medline]
Levade T, Moser HW, Fensom AH, Harzer K, Moser AB, Salvayre R (1995) Neurodegenerative course in ceramidase deficiency (Farber disease) correlates with the residual lysosomal ceramide turnover in cultured living patient cells. J Neurol Sci 134:108-114[Medline]
Moser HM (1997) Ceramidase deficiency: Farber lipogranulomatosis. In Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York, McGrawHill, 820-830
Nowaczyk MJM, Feigenbaum A, Silver MM, Callahan J, Levin A, Jay V (1996) Bone marrow involvement and obstructive jaundice in Farber lipogranulomatosis: clinical and autopsy report of a new case. J Inher Metab Dis 19:655-660[Medline]
Patel T, Gores GJ, Kaufmann SH (1996) The role of proteases during apoptosis. FASEB J 10:587-597
Qualman SJ, Moser HW, Valle D, Moser AE, Antonarakis SE, Boitnott JK, Zinkham WH (1987) Farber disease: pathologic diagnosis in sibs with phenotypic variability. Am J Med Genet Suppl 3:233-241[Medline]
Schwartz SM (1998) Cell death and caspase cascade. Circulation 97:227-229
Spicer SS (1987) Histochemistry in pathologic diagnosis. In Schwartz ME, ed. Clinical and Biochemical Analysis. Vol 22. New York, Marcel Dekker
Stassi G, Todaro M, Bucchieri F, Stoppacciaro A, Farina F, Zummo G, Testi R, De Maria R (1999) Fas/Fas ligand-driven T cell apoptosis as a consequence of ineffective thyroid immunoprivilege in Hashimoto's thyroiditis. J Immunol 162:263-267
Stassi G, Todaro M, De Maria R, Candore G, Cigna D, Caruso C, Galluzzo A, Giordano C (1997a) Defective expression of CD95 (FAS/APO-1) molecule suggests apoptosis impairment of T and B cells in HLA-B8, DR3-positive individuals. Hum Immunol 55:39-45[Medline]
Stassi G, De Maria R, Trucco G, Rudert W, Testi R, Galluzzo A, Giordano C, Trucco M (1997b) Nitric oxide primes pancreatic b-cells for Fas-mediated destruction in insulin-dependent diabetes mellitus. J Exp Med 186:1193-1200
Tanaka T, Takahashi K, Hakozaki H, Kimoto H, Suzuki Y (1979) Farber's disease (disseminated lipogranulomatosis) a pathological, histochemical and ultrastructural study. Acta Pathol Jpn 29:135-155[Medline]
Tepper AD, Boesende Cock JGR, de Vries E, Borst J, van Blitterswijk WJ (1997) CD95/Fas-induced ceramide formation proceed with slow kinetics and is not blocked by caspase-3/CCP32 inhibition. J Biol Chem 272:24308-24312
Testi R (1996) Sphingomyelin breakdown and cell fate. Trends Biol Sci 21:468-471
Thornberry NA, Lazebnik (1998) Caspases: enemies within. Science 281:1312-1316
Van Echten Deckert G, Klein A, Linke T, Heinemann T, Weisgerber J, Sandhoff K (1997) Turnover of endogenous ceramide in cultured normal and Farber fibroblasts. J Lipid Res 38:2569-2579[Abstract]
ZappatiniTommasi L, Dumontel C, Guibaud P, Girod C (1992) Farber disease: an ultrastructural study. Report of a case and review of the literature. Virchows Arch 420:281-290. [A]