The level of soluble A is elevated in the plasma and in the V 9/V 2 T cell culture supernatants of patients with active Behçet’s disease

A. Accardo-Palumbo1,3, A. Ferrante1, M. Cadelo1, F. Ciccia1, G. Parrinello2, L. Lipari1, A.R. Giardina1, M. Riili1, E. Giardina1, F. Dieli3, G. Triolo1

1Section of Rheumatology and Clinical Im- ABSTRACT in granule-mediated target munology and 2Section of Internal Medi- Objective. Gramzyme A (GrA) is a se - cell apoptosis (5). cine Department of Internal Medicine; and rine proteinase with -like activi - Circulating NK cells, and g/d T cells in 3 Section of General Pathology, Department ty that is released extracellularly dur - particular, participate in the early phas- of Biopathology, University of Palermo, ing the degranulation of cytotoxic cells. es of the immune response and are Italy. Among the cytotoxic cells, / T cells known to constitutively express per- Supported in part by a grant from the Min- participate in the early phases of the forin and granzymes in agreement with istero della Istruzione, della Università e della Ricerca (MIUR). Angelo Ferrante is immune response and are known to ex - their cytolytic pontential (6, 7). In con- a PhD student and recipient of a fellow- press perforin and granzymes constitu - trast, unstimulated CD3 CD8 CTL ex- ship from MIUR. tively in agreement with their cytolytic press only marginal levels of GrA m- Please address correspondence and re- pontential. RNA, but synthesis is rapidly induced print requests to: Prof. Giovanni Triolo, M e t h o d s . G r A activity was detected after IL-2 stimulation (8). In vitro stu- Cattedra di Reumatologia, Dipartimento using the synthetic substrate N- -ben - dies have shown that granzymes may Biomedico di Medicina Interna e Special- zyloxycarbonyl-L-lysine thiobenzyl es - be released extracellularly during cyto- istica, Istituto di Clinica Medica, Poli- ter in the plasma and supernatants of toxic cell degranulation (1). clinico Universitario, Piazza Cliniche 2, peripheral blood mononuclear cell cul - The immunopathogenesis of Behçet’s 90127 Palermo, Italy. E-mail: [email protected] tured in the presence of Dimethylallyl disease (BD) is believed to be T cell- Received on November 6, 2003; accepted pyrophosphate to obtain V 9/V 2 Tcell mediated. Cytotoxic T cells are consid- in revised form on April 14, 2004. expansion. ered to play a role in the development Clin Exp Rheumatol 2004; 22 (Suppl. 34): Re s u l t s . Significantly high levels of GrA of disease (9). Our recent data also S45-S49. were found in the serum and superna - point to a role of activated Vg9/Vd2 T tants of lymphocytes from patients with lymphocytes in the progression and © Copyright CLINICALAND EXPERIMENTAL RHEUMATOLOGY 2004. active Behçet’s disease cultured in the probably in the pathogenesis of the dis- pr esence of DMAPP. Levels were found ease (10). Key words: , Vg9/Vd2 T to be significantly lower after remis - Circulating levels of GrAhave not been cell, Behçet’s disease. sion. A positive correlation was observ - previously investigated in BD. In the ed between GrA levels in the superna - present study, we measured the amount tants and the V 9/V 2 T cell expansion of GrA in the serum of patients with factor. active and inactive BD. To extend stud- C o n c l u s i o n . These results stro n g l y ies on the role of Vg9/Vd2 T lympho- suggest that V 9/V 2 T cells are active cytes in the disease, we also investigat- participants in the pathogenesis of the ed GrA levels in the supernatants of disease through their degranulation lymphocytes from BD patients, cultur- and granzyme release. ed in the presence of Dymethylallyl py- rophosphate (DMAPP) to obtain Vg9/ Introduction Vd2 T cell expansion (10). Gramzyme A (GrA) is a serine protei- nase with trypsin-like activity that is re- Materials and methods leased extracellularly during the degra- Patients nulation of cytotoxic cells (1) Multiple 31 patients with Behçet’s disease (17 functions have been proposed for gran- males, 14 females, mean age 42 ± 24 zymes, including the degradation of ex- years), classified according to the Inter- tracellular matrix (2) and myelin basic national Study Group for Behçet’s dis- proteins in myelin membranes (3) and ease (11), were studied. At time of sam- the activation of pro- (4). Com- pling 18 patients were in the active and pelling evidence also supports a role for 13 were in the inactive stage. Patients

S-45 Granzyme Ain Behçet’s disease / A. Accardo-Palumbo et al. were considered to have active disease when at least two of the following cri- teria were present: recurrent aphthous ulceration, erythema nodosum, throm- bophlebitis, ocular and CNS involve- ment. Inactive patients included in this study were in complete remission (ab- sence of all signs and symptoms of dis- ease). Pathergy test positivity was pre- sent only in 27.7% of the patients in the active stage. The HLA-B51 aplotype was present in 16 of the patients. None of the case patients or control subjects had evidence of viral (EBV, CMV, HIV or HSV) or bacterial infection. In 6 of the active patients blood for serum and Fig. 1. Distribution of Granzyme A (GrA) serum levels in patients with active (BDA) and inactive lymphocyte studies was obtained after (BDI) Behçet’s disease, in patients with rheumatoid arthritis (RA), osteoarthritis (OA) and active tuber- the induction of remission. Remission culosis (Tbc) and in normal controls (NC). Results are expressed as mOD units at 405 nm. BDAvs BDI, p < 0.001; BDAvs NC, p < 0.001; BDAvs RA, NS; BDAvs OA, p < 0.001; BDAvs Tbc, in these patients was achieved by anti- p < 0.01; BDI vs OAvs Tbc, NS. T N Fa (Infliximab) therapy in 4 and high dose prednisone and cyclosporine in 2. All patients were using colchicine, tants may be either the result of GrAor RPMI 1640 supplemented with 10% an immunosuppressive agent such as GrK or both. The -like activity fetal calf serum (FCS, Euroclone), hep- cyclosporin (n=8), azathioprine (n=2), identified in the granules is, however, es 20 mM (Euroclone), 2 mM l-gluta- and/or low dose corticosteroids (n= 16). primarily attributable to GrA and both mine (Euroclone), penicilline/strepto- Twenty-one healthy volunteers (age GrA and GrK have been proposed to mycin 100 U/ml (Sigma) at 37°C, 0.5% range 21-54 years, mean 38 years) play a role in target cell death (13). CO2. For the expansion of Vg9/Vd2 T were enrolled as controls. In addition, cells (10), PBMC were cultured for 10 plasma from 5 patients with rheuma- Monoclonal antibodies and flow days in medium alone, or in the pres- toid arthritis, 8 patients with osteoarth- cytometry ence of Dimethylallyl pyrophosphate ritis and 6 patients with active tubercu- Monoclonal antibodies (mAbs) specif- (DMAPP; 0.5 mM, final concentration; losis were also investigated. Human ic for human surface anti-TCR Vd2 Sigma). After 72 hours, cultures were studies committee approval and indivi- FITC (PharMigen, San Diego, Ca) were supplemented with a 0.5 ml medium dual informed consent from each pa- used as followed: PBMC (106 in 100 ml containing 20 U/ml recombinant hu- tient were obtained. PBS with 1% heat-inactivated foetal man interleukin (IL-2; Genzyme, Cam- calf serum and 0.02% Na-azide) were bridge MA, USA). Every 72 hours 0.5 Esterase assay for GrA incubated at 4°C for 30 min. with anti- MLmedium was replaced with a 0.5 ml GrA activity was tested using the syn- TCRVd2 FITC conjugated mAb. After fresh medium containing 20 U/ml of thetic substrate N-a - b e n z y l o x y c a r- washing, the cells were suspended in IL-2. Results were expressed as expan- bonyl-L-lysine thiobenzyl ester (z-lys- PBS with 1% FCS and analysed on a sion factor (EF) (10). SBzl; Sigma, St Louis, USA). The este- FACScan flow cytometer (Becton rolytic activity against z-lys-SBzl was Dickinson, Mountain View, CA) by us- Statistical analysis assayed as previously described (12) ing forward scatter/side scatter gating Serum and supernatant GrA l e v e l s with minor modification. Briefly, 20 ml to select the lymphocyte population for from each group were compared using of supernatants (obtained at the 10th analysis. the Mann-Whitney U test. Levels for day of culture) or diluted (1:100 in each patient before and after remission PBS) sera were co-incubated with 35 Cell separation and expansion in vitro were compared using the student t test ml 1 mM z-lys-SBzl and 35 ml 1 mM of V 9/V 2 T lymphocytes. for paired data. The correlation be- 5,5 dithio-bis-(2 nitrobenzoic acid) Peripheral blood mononuclear cells tween GrA levels in supernatants and (Sigma). After incubation at 37°C for 2 (PBMC) were obtained from each indi- Vg9/Vd2 T cells expansion was anal- hours and 30 min. respectively, the ab- vidual by separating heparinized ve- ysed by the Spearman’s rank correla- sorbance at 405 nm was determined. nous blood on Ficoll (Euroclone, Weth- tion test. A p value < 0.05 was consid- Esterolytic activity of GrA was report- erby, Yorkshire, UK). The cells were ered to be significant. ed as mOD units. Because z-lys-SBzl washed in RPMI-1640 medium (Euro- can be hydrolyzed by granzyme K clone), and cultured in 24-well plates Results (GrK) as well as GrA, esterolytic activ- ( C o s t a r, Cambridge MA, USA) at a Serum levels of Gr A ity in the plasma or culture superna- concentration of 5 x 1 05 cells/ml in Serum GrA levels were significantly

S-46 Granzyme Ain Behçet’s disease / A. Accardo-Palumbo et al.

tuberculosis infection were 443 ± 3 7 (median 429; range, 396-497).

Supernatant levels of Gr A and V 9/V 2 T cell expansion GrA levels were determined in the su- pernatants of PBMC cultured in the presence of DMAPP to investigate the relationship between its release and the Vg9/Vd2 T cell expansion ability. GrAlevels were 818 ± 97 mOD units in the supernatants obtained from cultures Fig. 2. Serum level distribution of Granzyme A of active patients (n =6) (Fig.3). In in- Fig. 3. Level distribution of Granzyme A (GrA) (GrA) in patients with Behçet’s disease before active patient cultures (n = 5) GrA lev- in the supernatants of lymphocytes from patients (A) and after (B) the induction of clinical remis- els were 589 ±21, similar to those ob- with active (BDA) and inactive (BDI) Behçet’s sion. Results are expressed as mOD units at 405 disease or normal controls (NC) cultured in the nm. Avs B p < 0.01. tained in control cultures (n = 5; 550 ± presence of dimethylallyl pyrophosphate 30). The difference between levels in (DMAPP) to obtain the Vg9/Vd2 T cell expan- patients with active disease and con- sion. Results are expressed as mOD units at 405 higher in the active (746 ± 108 mOD trols or patients with inactive disease nm. BDA vs BDI, p < 0.001; BDA vs NC, p < 0.001; BDI vs NC, NS. units; median 777; range 435-888) than was statistically significant (p < 0.001) in the inactive patients (309±202; me- (Fig. 3). In the group of patients studied dian, 233; range, 85-692; p< 0.001) or before and after remission, levels were ology and pathogenesis of BD remain healthy controls (334 ± 203; median, 818±97 and 502±47 (p<0.001; Fig. 4) unknown. Various micro-organisms such 326; range, 47-659; p<0.001) (Fig.1). respectively. The Vg9/Vd2 expansion as streptococci and herpes simplex vi- GrA was also determined in a group of factor was 80 ±19 in active patients. In rus have been implicated as causative patients (n=6) before (765±169; medi- inactive patients and controls the ex- agents of DB in genetically susceptible an, 819; range, 435-888) and after re- pansion factor was 22±12 and 6±5, re- individuals (19-21). There is also evi- mission (361 ±260; median 230; range, spectively. Supernatant GrA levels in dence of immunological dysregulation, 125-692; p <0.01) (Fig.2). Serum lev- BD were significantly correlated with including neutrophil hyperfunction, au- els in patients with active rheumatoid the Vg9/Vd2 expansion factor obtained toimmune manifestations and several arthritis were similar (619 ± 108; medi- in the same cultures (n=11, r= 0.82, p< phenotypic and functional lymphocyte an, 625; range 498-741) to those ob- 0.01 ) (Fig.5). abnormalities, possibly resulting from tained in active BD. Low levels were complex interactions of genetic and found in the sera from patients with os- Discussion environmental factors (22-26). Most of teoarthritis (334±102; median, 325; Gramzyme A (GrA) is a serine protein- the immunological studies suggest a range, 185-481). Levels in patients with ase with trypsin-like activity that is re- central role for T cells in the patho- leased extracellularly during the degra- genesis of BD. Moreover, significant nulation of cytotoxic cells (14, 15). Cy- totoxic T cells have been shown to se- crete a significant portion of their syn- thesized granzymes following activa- tion (15); in particular, GrA is secreted by T lymphocytes in response to anti- gen or stimuli that mimic antigen (16). Analysis of highly purified lymphocyte population subsets suggests that GrAis endogenously expressed in NK and gd T cells, whereas a b T cells do not express high GrA levels (7). The con- stitutive expression GrA in gd T cells Fig. 4. Level distribution of Granzyme A (GrA) may be of significance for their poten- in the supernatants of lymphocytes from patients tial cytotoxic activity. Fig. 5. Correlation between the levels of Gran- with Behçet’s disease before (A) and after (B) B e h ç e t ’s disease is particularly fre- zyme A (GrA) in the supernatants of lympho- the induction of clinical remission cultured in the cytes from patients with Behçet’s disease, cul- presence of dimethylallyl pyrophosphate quent in countries along the Silk Road tured in the presence of dimethylallyl pyrophos- (DMAPP) to obtain the Vg9/Vd2 T cell expan- from the Mediterranean area to Japan, phate (DMAPP) to obtain the Vg9/Vd2T cell ex- sion. Results are expressed as mOD units at 405 and it is strongly associated with HLA- pansion, and the Vg9/Vd2 T cell expansion fac- nm. Avs B, p < 0.001. B51 and MIC-Agenes (17,18). The eti- tor (EF); (n=11, r=0.82, p<0.01).

S-47 Granzyme Ain Behçet’s disease / A. Accardo-Palumbo et al. proliferative responses of T cells to dition, GrA induces the production of munol 1998; 160: 3610-6. peptides derived from both mycobac- IL-6 and IL-8 in fibroblasts and epithe- 2. SIMON NM, SIMON HG, FRUTH U, EPPLEN J, MULLER-HERMELINK HK, KRAMER M D: terial and human 65-kD heat shock lial cells (30) and stimulates phago- Cloned cytolytic T- e ffector cells and their proteins has been demonstrated (27). cytic activity and IL-6, IL-8 and TNF- malignant variants produce an extracellular We and others have been recently pos- a production in monocytes (31). Our matrix degrading trypsin-like serine protei- tulated a role for gd T cells in the im- results do not indicate that increased nase. Immunol 1987; 60: 219-30. 3. VANGURI P, LEE E, HENKART P, SHIN ML: munopathogenesis of BD (10, 28). GrAlevels are specific for BD, high le- Hydrolysis of myelin basic protein in myelin In the present study we analysed the le- vels being obtained in other inflamma- membranes by granzymes of large granular vels of GrA in serum samples and in tory conditions (RA), but do suggest a lymphocytes. J Immunol 1993; 150: 2431-9. supernatants of PBMC from patients potential role for the GrA in the tissue 4. BRUNNER G, SIMON NM, KRAMER MD: Ac- tivation of pro-urokinase by the human T cell- with active or inactive BD cultured in damage of BD lesions. associated serine proteinase HUTSP-1. FEBS the presence of DMAPP, to obtain puri- GrA levels were observed in the serum Lett 1990; 260: 142-4. fied gd T cell populations (10). We uti- of active patients. GrA in the serum 5. NAKAJIMA H, PARK HL, HENKART PA: Sy- nergistic roles of granzymes A and B in medi- lized the BLT esterase assay to detect might be the consequence of degranu- ating target cell death by rat basophilic leuke- granzyme A a c t i v i t y. Other methods lation of gd T cells, but also of NK or mia mast cell tumors also expressing cyroly- have been recently described (western CTL. Impaired clearance of granzymes sin/perforin. J Exp Med 1995; 181: 1037-46. blotting and immunoassays), during a cytotoxic response may con- 6. KOIZUMI H, LIU CC, ZHENG LM, JOAG V, BAYNE NK, HOLOSHITZ J, YOUNG JD: Ex- but these detect the protein concentra- tribute, however, to an increase in ex- pression of perforin and serine esterases by tion and not enzymatic activity. Signifi- tracellular granzymes. Detectable lev- human gamma/delta T cells. J Exp Med 1991; cantly high levels of GrAwere found in els of extracellular GrA were also pre- 173: 409-502. the serum of patients with active dis- sent in the plasma of inactive patients 7. SAY E R S T J , B R O O K S A D , WA R D JM et al. : The restricted expression of granzyme M in ease, while levels in inactive patients and healthy donors. Since normal peri- human lymphocytes. J Immunol 2001; 166: and healthy controls were similar. Vg9/ pheral blood contains a small percent- 765-71. Vd2 cells from patients with active dis- age of activated granzyme-positive lym- 8. L I U CC, RAFII S, GRANELLI-PIPERNO A , ease, but not from inactive patients or phocytes, the levels in normal subjects TRAPANI JA, YOUNG JD: Perforin and serine esterase expression in stimulated human control individuals responded to may reflect the constitutive release of Tcells. J Exp Med 1989; 170: 2105-18. DMAPP in vitro with expansion. In- the proteases from these activated cells 9. YAMASHITA N, KANEOKA H, NANEKO S et creased GrA levels were found in the (32). In active patients, however, circu- al.: Role of gammadelta T lymphocytes in the culture supernatants of patients with lating GrAshould not only be the result development of Behçet’s disease. Clin Exp Immunol 1997; 107: 241-7. active disease but not in inactive pa- of the degranulation of CTL. CTL, in 10. TRIOLO G, ACCARDO-PALUMBO A, DIELI F tients. A positive correlation was also fact, are known to synthesize new pro- et al.: Vg9/Vd2 T lymphocytes in Italian pa- found between GrAlevels and the Vg9/ teins, some of which acquire the gran- tients with Behçet’s disease: Evidence for ex- Vd2 expansion factor. ule targeting signal and re-fill granules, pansion, and tumor necrosis factor receptor II and interleukin-12 receptor b1 expression in Vg9/Vd2 T lymphocytes from BD pa- and some of which are secreted via a active disease. Arthritis Res Ther 2003; 5: tients are activated (10). They may be non-granule pathway (15). In particu- 262-268. responsible for the development of in- lar, it has been observed that up to one- 11.INTERNATIONAL STUDY GROUP FOR BEHÇET’S DISEASE: Criteria for diagnosis of Behçet’s di- flammatory processes through cytokine third of the GrA secreted after T C R sease. Lancet 1990; 103: 177-84. production and the subsequent induc- cross-linking to release the lytic gran- 12. STENGER S, MAZZACCARO RJ, UJEMURA K tion of adhesion molecules, which per- ules is due to the newly synthesized pro- et al.: Differential effects of cytolytic T cell mit the accumulation of reactive T lym- teins secreted via the constitutive secre- subset on intracellular infection. S c i e n c e 1997; 276: 1684-7. phocytes at the site of inflammation. tory pathway; this protein is enzymati- 13. GA LV I N P J , SPA E N Y- D E K K I N G L H A , WA N G Involvement of gd T cells in the local cally active since it can be detected by B, SETH P,HACK CE,FROELICH CJ: Apopto- injury process has been also demon- cleavage of a synthetic substrate (15). sis induced granzyme B-glycosaminoglycan strated by their presence in the infiltrate The potential extracellular functions of complexes: Implications for granule-mediat- ed apoptosis in vivo. J Immunol 1999; 162: of mucosal ulcerations (29). We postul- the granzymes in disease have not been 5245-5350. ate the GrA secretion as a possible ef- extensively evaluated. Taken together, 14. V E L O T T I F, PA L M I E R I G , D ’ A M B R O S I O D , fector function of Vg9/Vd2 T cells in the elevated levels of GrA in serum PICCOLI M, FRATI L, SANTONI A: Differen- BD. and, in particular, in the Vg9/Vd2T cell tial expression of granzyme A and granzyme B proteases and their secretion by fresh rat GrAsecretion is induced by target cells supernatants of patients with active natural killer cells (NK) and lymphokine-acti- during the cytotoxic cell response. Cy- Behçet’s disease strongly suggest that vated killer cells with NK phenotype (LAC- tolytic cells have been reported to con- cytotoxic cells are active participants in NK). Eur J Immunol 1992; 22: 1049-53. stitutively secrete the GrA f o l l o w i n g the pathogenesis of the disease. 15. ISAAZ S, BAETZ K, OLSEN K, PODACK E, GRIFFITHS GM: Serial killing by cytotoxix T activation (15). gdT cells constitutively lymphocytes: T cell receptor triggers degra- express GrA(6,7). GrAhas been repor- References nulation, re-filling of the lytic granules and ted to degrade extracellular matrix pro- 1. SPA E N Y-DEKKING EHA, HANNA WL, W O L- secreation of lytic proteins via a non-granule BINK AM et al.: Extracellular granzymes A pathway. Eur J Immunol 1995; 25: 1071-9. teins in vitro (2) and myelin basic pro- and B in man: detection of native species dur- 16. TAKAYAMA H, STIKOVSKJ MV: Antigen re- teins in myelin membranes (3). In ad- ing CTLresponses in vitro and in vivo. J Im- c e p t o r-regulated exocytosis in cytotoxic T

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