Alteration of peripheral blood lymphocyte subsets in acute pancreatitis

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1 PO Box 2345, Beijing 123, Chin World J Gstroenterol 26 September 7; 12(33): World Journl of Gstroenterology ISSN wjg@wjgnet.om 26 The WJG Press. All rights reserved. CLINICAL RESEARCH Altertion of peripherl blood lymphoyte subsets in ute pnretitis Miroslw Pietruzuk, Milen I Dbrowsk, Urszul Wereszzynsk-Siemitkowsk, Andrzej Dbrowski Miroslw Pietruzuk, Milen I Dbrowsk, Deprtment of Hemtologil Dignostis, Medil University of Bilystok, Polnd Urszul Wereszzynsk-Siemitkowsk, Andrzej Dbrowski, Deprtment of Gstroenterology nd Internl Mediine, Medil University of Bilystok, Polnd Supported by the Stte Committee for Sientifi Reserh (KBN) grnt 4 PO5B Correspondene to: Professor Andrzej Dbrowski, Deprtment of Gstroenterology nd Internl Mediine, Medil University of Bilystok, Sklodowsk-Curie 24A, Bilystok , Polnd. dbrows@mb.edu.pl Telephone: Fx: Reeived: Aepted: Abstrt AIM: To evlute peripherl blood lymphoyte subsets in ptients with ute pnretitis (AP). METHODS: Twenty ptients with mild AP () nd 15 with severe AP () were inluded in our study. Peripherl blood lymphoytes were exmined t d 1-3, 5, 1 nd 3 by mens of flow ytometry. RESULTS: A signifint depletion of irulting lymphoytes ws found in AP. In the erly AP, the mgnitude of depletion ws similr for T- nd B- lymphoytes. In the lte ourse of, B-lymphoytes were muh more depleted thn T-lymphoytes. At d 1, strong shift in the CD7+/CD19+ rtio impliting predominne of T- over B-lymphoytes in ws found. Among T-lymphoytes, the signifint depletion of the CD4+ popultion ws observed in nd, while CD8+ ells were in the norml rnge. Lymphoytes were found to strongly express tivtion mrkers: CD69, CD25, CD28, CD38 nd CD122. Serum interleukin-2 (IL-2), IL-4, IL-5, IL-1, interferon-γ (IFN-γ) nd tumor nerosis ftor-α (TNF-α) levels were signifintly inresed in both forms of AP. The mgnitude of elevtion of ytokines known to be produed by Th2 ws muh higher thn ytokines produed by Th1 ells. CONCLUSION: AP in humns is hrterized by signifint redution of peripherl blood T- nd B-lymphoytes. 26 The WJG Press. All rights reserved. Key words: Aute pnretitis; Flow ytometry; Inflmmtory ytokines; Lymphoytes Pietruzuk M, Dbrowsk MI, Wereszzynsk-Siemitkowsk U, Dbrowski A. Altertion of peripherl blood lymphoyte subsets in ute pnretitis. World J Gstroenterol 26; 12(33): INTRODUCTION Exessive leukoyte tivtion with ytokinemi represents one of the most importnt mehnisms of inresed mortlity in erly ute pnretitis (AP). It hs been hypothesized tht ftl pnretitis is onsequene of exessive leukoyti phgoyte stimultion provoked by severe trum or persistent injury due to n gent noxious to the pnres [1]. Lymphoytes nd inflmmtory meditors relesed by these ells re one of the most potent regultors of leukoyti phgoytes. It hs been suggested tht T- nd B- lymphoytes tivtion is key ftor in the modultion of the inflmmtory retion in different diseses, inluding AP [2]. The role of different inflmmtory meditors in AP hs lredy been extensively studied, while the role of lymphoyte tivtion nd its reltion to disese severity in humns re still poorly understood [3-6]. Few studies hve demonstrted the redution of totl peripherl lymphoytes s well s CD4+, CD8+, CD3+DR- nd CD3-DR+ lymphoyte subsets in erly severe AP [7-1]. In the most reent study, T ell tivtion hs been reported in mild ute pnretitis [11]. Another study showed tht tretment with high-dose vitmin C prtilly restored depleted peripherl blood CD4+ ells nd inresed the rtio of CD4+/CD8+ ells [12]. Under the norml or pthologi onditions, CD4+ T helper (Th) lymphoytes polrize into two mjor subsets: Th1 nd Th2. Both environmentl nd geneti ftors t in onert to determine the Th1 nd Th2 polriztion. The olletive dt vilble so fr re not suffiient to determine the role of speifi Th ells subsets in ute pnretitis in humns. In this study, we, therefore, imed to provide brod nd omplex evlution of peripherl blood lymphoyte subsets in ptients with AP.

2 Pietruzuk M et l. Lymphoyte subsets in pnretitis 5345 MATERIALS AND METHODS Clinil evlution Thirty five ptients (16 women nd 19 men; ge yers, medin 56 yers) with AP were prospetively inluded into our study. In ll ptients, the time between the bdominl pin onset nd dmission to the hospitl ws not longer thn 48 h. The ontrol group omprised of 15 helthy volunteers (7 women nd 8 men; ge yers, medin 41 yers) without the history of reent inflmmtory disese. The dignosis ws mde on the bsis of history onsistent with AP nd serum mylse tivity > 3 times the upper limit of norml rnge (2-9 U/L). For ll the ptients, dditionl biohemil nd imging (ultrsound nd omputed tomogrphy) tests hve been done to onfirm the dignosis s well s to determine the etiology nd severity of AP ording to Rnson s [13] nd Blthzr s [14] riteri supplemented by serum C-retive protein (CRP) onentrtion mesurements. At d 2-3, CRP onentrtions higher thn 15 mg/l were onsidered s inditive of. Using these riteri, our ptients were divided into mild AP (n = 2) nd severe AP (n = 15) (Tble 1). Among the ptients with, the following systemi omplitions were found: pulmonry in 8 ptients, irultory in 3, renl in 3, ogultion disorders in 1 nd septi omplitions in 1 ptient. Lol omplitions, suh s ute fluid olletions in 1 ptients, infeted nerosis in 1 nd pnreti bsess in 1 ptient, were found. By etiology, 18 ptients presented biliry pnretitis, 16 loholi pnretitis nd 1 idiopthi pnretitis. In the ourse of hospitliztion, 2 ptients with severe AP died. Medil University of Bilystok Ethil Committee pprovl hd been grnted for performing this study. Tretment In ptients with, in ddition to stndrd tretment, prophylti therpy with ntibioti (meropenem 5 mg tid for 1-21 d) nd enterl nutrition hve been implemented sine the d 2-3 of the disese. Lbortory methods EDTA-ntiogulted blood smples were tken t dmission (d 1), on d 2, 3, 5, 1 nd 3. Blood smples were drwn from the nteubitl vein. The bsolute number of leukoytes (grnuloytes, lymphoytes, monoytes) ws estimted with hemtologil nlyzer Advi 12 (Byer). Surfe lymphoyte ntigens (CD) were ssyed by the diret fluoresene method for whole blood, using flow ytometer (EPICS XL, Coulter) nd double stining (FITC/PE) monolonl ntibodies (Beton Dikinson). Briefly, 1 μl smples of whole blood were inubted with 5 μl of respetive monolonl ntibody solution. After 3 min of inubtion in the drk t 4, erythroytes were lysed, while leukoytes were fixed, stbilized (ImmunoPrep, Coulter) nd nlysed by EPICX XL. The use of gte-hek [CD45-FITC/CD14-PE (BD)] llowed the division of the leukoytes popultion into grnuloytes, lymphoytes nd monoytes. Mthed lbelled nti-idiotype ntibodies were used s negtive ontrols (IgG1-FITC/IgG1-PE). Tble 1 Clinil hrteriztion of studied ptients with mild AP nd severe AP Mild AP Severe AP P n = 2 n = 15 vs (Mnn-Whitney U test) Etiology Biliry 9 (45.%) 9 (6.%) Alohol 1 (5.%) 6 (4.%) Idiopthi 1 (5.%) (%) Medin rnson 1 (-2) 4 (2-8) P <.1 sore (rnge) Blthzr sore A 6 (3.%) B 14 (7.%) C 2 (13.3%) D 5 (33.3%) E 8 (53.3%) CRP (mg/l) medin (rnge) D ( ) 216. ( ) P <.1 D ( ) ( ) P <.1 Popultions nd subpopultions of lymphoytes Popultions nd subpopultions of lymphoytes were evluted using the following differentition ntigens: CD19-/CD7+ (T-lymphoytes), CD3+/CD4+ (Thlymphoytes), CD3+/CD8+ (ytotoxi T-lymphoytes) nd CD19+/CD7- (B-lymphoytes). Ativtion mrkers of T-lymphoytes The following CD ntigens were exmined: CD3/CD69, CD7/CD122 nd CD8/CD38, CD8/CD28. Ativtion mrkers of B-lymphoytes CD19/CD122 expression ws determined in B-lymphoytes. Determintion of poptosis Cell preprtion: Mononuler ells were isolted by density grdient onentrtion on Histopque 177 (Sigm). Highly purified mononuler ells (bout 98%, by Advi 12) were obtined. The ells were subsequently suspended in tubes with phosphte-buffered sline. Determintion of spontneous poptosis: Apoptosis ws determined using Annexin V-Fluos nd propidium iodide double stining. Apoptosis is ompnied by loss of membrne phospholipid symmetry, resulting in the exposure of phosphtidylserine t the surfe of the ell. Quntittive mesurement of phosphtidylserine exposure ws possible using the binding of fluoresein isothioynte-lbelled nnexin V to phosphtidylserine. For the nnexin V ssy, ells were inubted for 1 h t 37 in 2 ml of buffer ontining FITC-lbelled nnexin V nd propidium iodide (PI) nd line speifi mrkers (CD3, CD19, CD33, CD14, PE). Flow ytometri nlysis ws performed by Coulter EPICS XL. Determintion of stimulted poptosis Determintion of stimulted poptosis ws rried out s previously desribed [15]. Isolted lymphoytes were inubted in six-well pltes (Flon, Beton Dikinson)

3 5346 ISSN CN / R World J Gstroenterol September 7, 26 Volume 12 Number 33 with RPMI 164 medium (GIBCO) without fetl bovine serum t 37 in humidified tmosphere ontining 5 ml/l CO2. After 24 h of inubtion, ells were stined with dye mixture (1 μmol/l ridine ornge nd 1 μmol/l ethidium bromide; Sigm) prepred in phosphtebuffered sline (PBS). Two-hundred ells per smple were exmined by fluoresene mirosopy, nd the ells were hrterized ording to the following riteri: (1) ntive ells-fine retiulr pttern of green stin hromtin; (2) neroti ells-bright ornge stin hromtin; nd (3) poptoti ells-green stin hromtin whih ws highly ondensed nd uniformly stined by ridine ornge. A B ,,, Control ,,, (d) Determintion of ytokines produed by Th1/Th2 subsets The BD Humn Th1/Th2 Cytokine CBA kit ws used to mesure IL-2, IL-4, IL-5, IL-1, TNF-α nd IFN-γ levels in serum smples. The smples diluted with the pproprite volume of Assy Diluent were trnsferred into the ssy tubes ontining pture beds (overed with respetive ytokine ntibody) nd PE detetion regent. The stndrd urve for eh ytokine ws defined for onentrtions from 2-5 ng/l. The ssy ws performed using FACSClibur (BD) flow ytometer. Sttistil nlysis Sttistil nlysis ws performed using the non-prmetri Mnn-Whitney s U test. P vlues less thn.5 were onsidered sttistilly signifint. Results were expressed s men ± SD. RESULTS At d 1, signifint derese of totl lymphoyte number to 63% nd 39% of the helthy ontrol ws found in ptients with nd, respetively (Figure 1A). In the ourse of the disese, lymphoyte number grdully inresed, rehing 79% nd 97% of the ontrol in nd t d 1, respetively. At d 3, lymphoyte number fell gin to 65% nd 42% of the ontrol, respetively, in nd. Anlysis of CD7 nd CD19 moleule expression showed tht T-lymphoyte (CD7+) popultion ws espeilly redued in the erly ourse of (Figure 1B). Therefter, it ws onstntly inresing, returned to norml t d 1 nd deresed gin to 46% of the ontrol t d 3. In, depletion of T-lymphoyte popultion ws less evident (Figure 1B). Some redution of B-lymphoyte (CD19+) number ws found in the erly, while mrked depletion to 36% nd 34% of the ontrol ws notied t d 1 nd 3, respetively (Figure 1C). In, the derese of B-lymphoyte number ws not so persistent. Two mjor subsets of T-lymphoytes re represented by CD4+ nd CD8+ ells, lso known s T4-helper ells nd T8-ells or ytotoxi T-lymphoytes (CTLs), respetively. At d 1, the number of irulting CD4+ ells signifintly dropped in both nd to 68% nd 45% of the ontrol, respetively (Figure 1D). In, t d 5 nd 1, it returned to the rnge of norml level, while in it remined signifintly depleted until d 3. The number of irulting CD8+ ells remined in the rnge C D E F G Control (d) of the norml level in the erly, inluding the d 1 (Figure 1E). At d 3, it deresed to 73% of the ontrol. Control (d),,, Control (d) Control (d),,, Control (d) Figure 1 Peripherl blood lymphoytes in ptients with AP. A: Totl ount; B: CD7+; C: CD19+; D: CD4+; E: CD8+; F: CD69+; G: CD25+. Results re expressed s men ± SD. P <.5 vs ontrol; P <.5 vs mild AP t the sme dy of observtion. Control (d),,,,,,,,,,

4 Pietruzuk M et l. Lymphoyte subsets in pnretitis 5347 Tble 2 Peripherl blood lymphoyte CD7+/CD19+ nd CD4+/CD8+ rtio in ptients with AP Dys Control CD7+/CD19+ CD4+/CD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.5.7 ± ± ± ± ± ± ± ± ±.6.9 ± ±.5 P <.5 vs ontrol; P <.5 vs mild AP t the sme dy of observtion. A , Control (d) A , Control (d), B Control (d) B Control (d) Figure 2 Peripherl blood T-lymphoytes (CD7+) (A) nd B-lymphoytes (CD19+) (B) expressing CD122 in ptients with AP. Results re expressed s men ± SD. P <.5 vs ontrol; P <.5 vs mild AP t the sme dy of observtion. Figure 3 Peripherl blood CD8+ ells expressing CD38 (A) nd CD28 (B) in ptients with AP. Results re expressed s men ± SD. P <.5 vs ontrol; P <.5 vs mild AP t the sme dy of observtion. At d 1 of, CD8+ ells were mrkedly depleted to 47% of the ontrol nd remined below norml level until d 5. CD8+ returned to norml level t d 1, nd depleted gin to 47% of the ontrol t d 3. In ptients with both nd, some flututions of lymphoyte T/B (CD7+/CD19+) rtio were observed (Tble 2). At d 1, the rtio ws mrkedly inresed beuse the T-lymphoyte number ws lose to norml nd the signifintly diminished B-lymphoyte level. In nd, the lowest CD4+/CD8+ ells rtio ws found t d 3 nd 1, respetively (Tble 2). Peripherl blood lymphoytes in both nd ptients showed drmti inrese of intrellulr signling mnifested by CD69 expression. At d 1, it ws elevted 6.-fold nd 4.6-fold in nd, respetively (Figure 1F). At d 1, CD69 expression in peripherl blood lymphoytes rehed mximum (11.2-fold over the ontrol level) in nd then drmtilly delined to 52% nd 4% of the helthy ontrol in nd, respetively. CD25/IL-2Rα expression, known s the lymphoyte prolifertion mrker, ws elevted espeilly in (Figure 1G). At d 1 nd 1, it inresed to 3.3-fold nd 4.2-fold over the ontrol, respetively. In, mrked elevtion of CD25/IL-2Rα expressing lymphoytes (4.5-fold over the ontrol) ws found t the d 1. At d 3, the number of the lymphoyte subpopultion signifintly deresed to 63% in nd to 47% in. The number of peripherl blood T-lymphoytes expressing CD122 shrply inresed in ptients with (Figure 2A). At d 1, it inresed lmost 2-fold ompred to the ontrol, nd remined highly elevted until d 5. At the d 1, the number of lymphoytes signifintly diminished nd t d 3, rehed 5% of the ontrol level. In the ourse of, the number of T-lymphoytes expressing CD122 did not signifintly hnge. However, t d 3, it dropped to 33% of the norml level. The number of peripherl blood B-lymphoytes with the sme mrker of tivtion ws signifintly elevted in both nd ptients (Figure 2B). Interestingly, onerning CD122 expression, the highest level of B-lymphoyte tivtion, 6- nd 7-fold were found in nd t the d 3, respetively. Figure 3 shows the pttern of tivtion of T-lymphoyte CD8+ subset. In, the number of ells expressing CD38 inresed to 2.6% of the ontrol level t d 1, rehed the mximum level of 33.9% t d 3 nd remined elevted t 2.3% t d 3 (Figure 3A). In, the opposite trend ws noted with signifint diminishing of CD8+CD38+ lymphoytes to 45% of the ontrol t d 1. It returned to norml level t d 3, while strong temporry elevtion to 49.7% of the ontrol t d 1 ws noted. Conerning CD28 expression, CD8+ T-lymphoyte tivtion ws the strongest observed in ptients (Figure 3B). At d 1, it rehed 28.8% of the ontrol, remined t the similr level until d 3 nd then slightly deresed to 19.2% of the ontrol t d 3. In, it osillted round norml

5 5348 ISSN CN / R World J Gstroenterol September 7, 26 Volume 12 Number 33 A B ,, Control (d),,,,,, Control (d) Tble 3 Apoptosis nd nerosis rte in lymphoytes isolted from ptients with AP Cultured with serum Cultured without serum (% of totl) (% of totl) Apoptosis Living Nerosis Apoptosis Living Nerosis Control 2 ± 1 96 ± 2 1 ± 1 23 ± 2 1 ± 3 68 ± 3 3 ± 1 97 ± 1 1 ± 1 5 ± 6 17 ± 6 33 ± 4 3 ± 1 96 ± 2 2 ± 2 5 ± 6 15 ± 4 36 ± 5 P <.5 vs ontrol. Tble 4 Serum onentrtion of ytokines produed by Th1 lymphoyte subpopultion in ptients with AP C 25 2 Dys Control ,,,,,, Control (d) IL-2 2 ± 6 18 ± 8 18 ± 7 12 ± 7 8 ± ± 5.4 (ng/l) 21 ± 1 24 ± ± ± 9 9 ± 5 IFN-γ 45 ± ± ± ± ± 5 6. ± 5.7 (ng/l) 258 ± ± 23 4 ± ± 1 16 ± 6 TNF- 2.9 ± ± ±.9.9 ±.7 1. ± ± 2.4 (ng/l) 3.2 ± ± ± ± ±.9 Figure 4 Mrkers of lymphoyte poptosis in ptients with AP. Peripherl blood lymphoytes expressing CD95 (A), CD3+ lymphoytes expressing Annexin V (B) nd CD19+ lymphoytes expressing Annexin V (C). Results re expressed s men ± SD. P <.5 vs ontrol; P <.5 vs mild AP t the sme dy of observtion. level until d 5, inresed to 23.6% of the ontrol t d 1, nd remined slightly elevted t d 3. Expression of CD95/Fs-R/Apo-1 reeptor protein, whih plys n importnt role in poptosis initition, ws enhned in erly nd rehed mximum (18.8% of the ontrol) t d 3 (Figure 4A). It remined slightly elevted throughout the ourse of the disese. In, the number of lymphoytes expressing CD95/Fs-R/Apo-1 slightly deresed to 7.6% of the ontrol t the d 1 nd then onstntly rised up to 26.8% t d 1. Therefter, it returned to the ontrol level t the d 3. At d 1, the number of peripherl blood T-lymphoytes tht stined for nnexin V (AnnV+CD3+) signifintly deresed in nd to 49% nd 17% of the norml level, respetively (Figure 4B). Therefter, it grdully inresed, nd in slightly exeeded norml level (12.2% of ontrol) t d 5. In, the number of AnnV+CD3+ lymphoytes remined diminished during the ourse of whole observtion. The number of peripherl blood B-lymphoytes tht stined for nnexin V (AnnV+CD19+) strted to derese t d 1 in both forms of AP, nd t d 1, rehed the minimum vlue, i.e. 3% nd 1% of the norml level in nd, respetively (Figure 4C). Tble 3 shows the vibility of lymphoytes isolted from peripherl blood of ptients with pnretitis. In both forms of pnretitis, in nonstimulted ells, the rtes of poptosis nd nerosis were t similr levels s in the ontrol. Stimulted ells from the helthy ontrol died predominntly by nerosis. However, in stimulted ells of ptients with pnretitis, lymphoytes died predominntly by poptosis, without P <.5 vs ontrol; P <.5 vs mild AP t the sme dy of observtion. Tble 5 Serum onentrtion of ytokines produed by Th2 lymphoyte subpopultion in ptients with AP Dys Control IL-4 6. ± ± ± 6 79 ± ± 43 6 ± 33 (ng/l) 198 ± ± ± ± ± 35 IL ± ± 9 29 ± 9 17 ± 7 52 ± 23 4 ± 3 (ng/l) 23 ± 7 4 ± ± 7 39 ± 19 3 ± 3 IL-1 4 ± 27 5 ± ± ± ± ± 1.6 (ng/l) 52 ± ± ± 36 5 ± ± 31 P <.5 vs ontrol; P <.5 vs Mild AP t the sme dy of observtion. showing ny signifint differene between nd. We mesured serum onentrtions of ytokines produed by Th1 nd Th2 lymphoytes in the first 1 dys of the disese (Tbles 4, 5). IL-2, IFN-γ nd TNF-α represent the pttern hrteristi for Th1, while IL-4, IL-5 nd IL-1 re produed by Th2. IL-2 nd IFN-γ levels were muh higher in thn in, while TNF-α ws slightly elevted in both forms of pnretitis (Tble 4). At d 1, we observed huge inrese by 27-fold nd 33-fold in the IL-4 level in nd, respetively (Tble 5). It remined highly elevted in the whole erly ourse of pnretitis. IL-5 ws signifintly inresed in both forms of pnretitis during the first 5 d of observtion. However, it returned to norml level t d 1. A remrkble inrese of IL-1 ws noted in both forms of pnretitis. The pek, 36-fold nd 44-fold elevtion, ws observed in nd t d 2, respetively (Tble 5).

6 Pietruzuk M et l. Lymphoyte subsets in pnretitis 5349 DISCUSSION Altertion of the immune system is one of the mjor mehnisms responsible for erly nd lte mortlity in severe AP. Exessive inflmmtory retion, known s systemi inflmmtory response syndrome (SIRS), is onsidered s the leding use of deth in erly AP [4-6]. The role of lymphoytes in this phenomenon hs been prtly studied nd the knowledge of the mehnisms in humns is still inomplete [7-11,16]. Our study provides, probbly for the first time, brod nd omplex nlysis of peripherl blood lymphoyte subsets with referene to different time points nd different severity forms of ute pnretitis. In greement with the previous studies [7-11,16], we lso found signifint depletion of irulting lymphoytes whih ws muh more profound in the severe form of pnretitis. In the erly period of 1-5 d of the disese, the mgnitude of peripherl depletion ws similr for T- (CD7+) nd B- (CD19+) lymphoytes. However, in the seond week nd t d 3 of, B-lymphoytes were found to be prtiulrly depleted. Conerning the reltions between these subsets of lymphoytes, the middle of the seond week (d 1) ws ritil beuse of the strong predominne of T- over B-lymphoytes in. Depletion of the CD19+ lymphoyte subset in erly ute pnretitis hs lso been reently reported [16]. Among T-lymphoytes, t d 1 nd 3 of s well s t d 1 of, we observed signifint depletion of the CD4+ popultion, while CD8+ ells were in the norml rnge. It reted temporry imblne in the ell rtio with the pprent prevlene of CD8+ over the CD4+ ells. CD8+ ells re known s ytotoxi T-lymphoytes (CTL) nd some of them differentite into T8-suppressor ells. Signifint depletion of peripherl blood CD4+ nd CD8+ subpopultions of T-lymphoytes in the ourse of ute pnretitis hs previously been reported [7-9,12,16]. However, observtions mde by the others were restrited mostly to one or up to three different time periods of pnretitis. The spetrum of ytokines known to modulte lymphoyte differentition nd funtion (IL-2, IFN-γ, TNF-α, IL-4, IL-5 nd IL-1), evluted in the erly ourse of pnretitis, showed ll of them signifintly inresed in both forms of AP, however, the highest vlues were observed in. Previous studies showed the elevted IL-2, TNF-α nd IL-1 levels in humn AP [11,12,17,18]. To our knowledge, no dt re vilble in literture on serum levels of IFN-γ, IL-4 nd IL-5 in humn AP. In our study, the mgnitude of elevtion of ytokines known to be produed by Th2 ells, nmely IL-4, IL-5 nd IL-1, ws muh higher thn ytokines produed by Th1 ells, represented by IL-2 nd IFN-γ. This finding suggests tht in the ourse of AP, Th1 subpopultion of CD4+ ells is suppressed more strongly thn Th2. Erly IL-4 expression during n immune response is ritil for determining the development of Th2 ells [19]. Cytokines produed by Th2 ells enble tivted B-lymphoytes to proliferte, stimulte tivted B-lymphoytes to synthesize nd serete ntibodies, promote the differentition of B-lymphoytes into ntibody-sereting plsm ells, nd enble ntibodyproduing ells to swith the lss of ntibodies being produed [2]. In our study, B-lymphoyte tivtion ws the strongest t d 3, while T-lymphoyte tivtion remined norml or even below the ontrol level. This pttern of lymphoyte tivtion might be the onsequene of shift from Th1 to Th2 in ptients with AP. Th1 nd Th2 ells represent polrized forms of the CD4+ Th ell-medited immune response. Th1 ells produe IL-2, IFN-γ nd TNF-α whih ooperte with B-ells for the prodution of ntibodies, tivte phgoyti ells nd CD8+ T-ells, thus promoting ell-medited immunity nd ytotoxi T-ell responses [21]. In ontrst, ytokines relesed by Th2 ells (IL-4, IL-5, IL-9 nd IL-13) indue B-ells to produe high mounts of IgG4 nd IgE in humns, promote the differentition nd growth of mst ells nd eosinophils, nd inhibit severl phgoyti funtions [21]. Of note, while IL-4 inhibits the development of Th1 ells, IFN-γ inhibits the development of Th2 ells [21]. Treg ells re highly heterogeneous fmily, whih inludes type 3 Th (Th3) ells, T regultory 1 (Tr1) ells, nd CD4+ CD25+ T ells. Interestingly, Tr1 ells re minly ble to produe IL-1. Therefore, the huge elevtion of serum IL-1 level found in our study my suggest tht Tr1 ells re espeilly tive in the ourse of AP. In our study, signifint tivtion of lymphoytes ws observed, whih ws shown by strong expression of CD69, CD25, CD28, CD38 nd CD122 on T-lymphoytes s well s CD122 on B-lymphoytes. The surfe reeptors CD69 nd the IL-2 reeptor (CD25) re erly mrkers of tivtion. Inresed expression of CD69 s well s CD25 on CD3+, CD4+ nd CD8+ ells hs reently been reported in mild AP [11]. It hs lso been reported tht the number of B-lymphoytes (CD19+) expressing CD69+ ws signifintly lower in ptients with severe pnretitis thn in ptients with mild pnretitis [16]. Consequently, the onlusion hs been mde tht ptients with severe pnretitis show impired erly tivtion of peripherl CD19+ ells. Interestingly, in our study, evlution of CD19+ ells expressing CD122, whih is nother mrker of lymphoyte tivtion, showed signifint tivtion of the B-lymphoyte subset bering this mrker. These observtions my suggest tht the CD19+ ell subset is heterogeneous nd the subsets of these ells ret in different mnner in the ourse of pnretitis. Mrked tivtion of different subsets of lymphoytes my explin why we hve observed strong elevtion of different ytokines in peripherl blood despite signifint derese in the number of irulting lymphoytes. The question remins, however, wht ws the reson for signifint depletion of peripherl blood lymphoytes in pnretitis. One of the possible resons is strong migrtion of tivted lymphoytes to the site of inflmmtion, inluding the pnres nd other tissues like lungs or kidneys, s prt of SIRS [22]. Another possible explntion is n exessive elimintion of lymphoytes by poptosis. Expression of CD95, known s Apo-1, Fs or deth reeptor on lymphoytes ws inresed in erly nd in t d 1. Surprisingly, the expression of nnexin V, whih is mrker of ongoing poptosis, ws deresed in erly nd lte on both T- nd B-lymphoytes. In the lte period of, nnexin V expression on T-lymphoytes ws in the norml rnge,

7 535 ISSN CN / R World J Gstroenterol September 7, 26 Volume 12 Number 33 while it ws deresed on B-lymphoytes. This finding is in greement with our observtion of inresed tivtion of B-lymphoytes in the lte ourse of pnretitis. It is known tht stimultion by ftors induing ell prolifertion my inhibit poptosis [23]. This phenomenon is the bsis of the method evluting ell suseptibility to poptosis [15]. Using this in vitro method, we found tht lymphoytes from ptients with pnretitis were primed to poptosis. Similr results of in vitro inubtion of lymphoytes from ptients with hve previously been reported [1]. Tken together, peripherl blood lymphoyte depletion in ute pnretitis my result from both exessive poptosis nd migrtion to the site of inflmmtion. Tissue dmge used not only by severe AP but lso by trumti inidents, like severe burns, ident trum, mjor surgil interventions or sepsis, indues ommensurte with the severity of dmge (dmge lod), geneti ftors (gene polymorphism), the generl ondition of the host nd the type of ntigens (ntigeni lod), both lol nd systemi relese of pro-inflmmtory ytokines nd phospholipids [24-26]. Polymorphonuler leukoytes, monoytes, tissue mrophges, lymphoytes, nturl killer ells, nd prenhyml ells re involved in omplex network of the host defense response. An overwhelming pro-inflmmtory response (hyperinflmmtion) leds to the linil mnifesttions of SIRS nd finlly to host defense filure expressed by multiple orgn dysfuntion syndrome (MODS) or multiple orgn filure (MOF). The up-regultion of pro-inflmmtory ftors, suh s TNF, IL-1 nd IL-6, observed during the SIRS phse n be followed by seond response tht involves down-regultion of IFN-γ nd inrese in ntiinflmmtory ytokines, suh s IL-1 nd trnsforming growth ftor-β. This ounter-regultory phenomenon is lled the ompenstory nti-inflmmtory response syndrome (CARS) [24]. Aside from ytokine profiles, ritil injury nd sepsis re lso orrelted with dysfuntion of mny immune ells. After severe injury, CD4+ T-ell differentition into Th phenotypes is ltered nd there is n erly expression of Th1 ytokines (IL-12, IFN-γ), followed 24 to 72 h lter by predominne of the Th2 ytokine (IL-4) nd depression in the prodution of IL-2 nd IFN-γ [24]. It hs been found tht severe AP, burns, ident trum, mjor surgil interventions or sepsis re ssoited with signifint derese in totl systemi lymphoyte ounts, inluding both CD4+ nd CD8+ ells. The development of immunosuppression in subjets suffering from trumti events is often ssoited with elevtion of IL-1 nd the shift of the Th1/Th2 blne towrds Th2 response [24,26]. The mjority of dt bout lymphopeni in trum-relted ellulr immune defets refer to T-ells with snt informtion bout B-ells. Lung nd/or kidney filure my tke ple in the ourse of speifi diseses ffeting these orgns or my be prt of MOF or MODS resulting from tissue dmging events, inluding. It hs been found tht peripherl blood B-ells, but not CD4+ nd CD8+, were signifintly lower in uremi ptients. This phenomenon my be prtilly ttributed to n inresed suseptibility to poptosis ssoited with deresed expression of Bl-2 [27]. On the ontrry, T lymphopeni with norml ounts of peripherl blood B-ells hs reently been reported in end-stge renl disese [28,29]. Ex vivo evlution of T-ells showed n inresed number of nnexin V nd CD95 (Fs)-positive T-ells, suggesting tht poptosis my be responsible for exessive elimintion of this lymphoyte subpopultion [28]. A progressive derese in renl funtion ssoited with tivtion nd seletive loss of nïve CD4+ nd CD8+ T-ells s well s CD4+ entrl memory ells hs reently been reported. These hnges my ontribute to the linil phenomen of the uremissoited immune defet in ptients with hroni renl disese [29]. Aute respirtory distress syndrome (ARDS), sudden, life-thretening lung filure, n omplite the ourse of severe AP nd other diseses with ritil tissue dmge. Severe lymphopeni hs reently been reported in ptients with severe ute respirtory syndrome (SARS) who developed ARDS [3]. ARDS ourring in renl trnsplnttion ptients with pneumoni is ompnied by signifint derese in blood CD4+ nd CD8+ T ells. In these ptients, the reovery of the disese met the reovery ourse of their immune system [31]. In onlusion, the vilble dt indite tht lymphopeni used by the derese of different lymphoyte subpopultions is hrterized not only for severe AP but lso for other diseses with severe tissue dmge. However, with the exeption of our study, the vilble dt provide mostly the informtion bout single or just few representtives of lymphoyte subpopultions in speifi disese. Further studies involving brod spetr of immune proesses regultion re neessry in order to better understnd the omplex phenomenon of immunity disorders in diseses with ritil tissue dmge. In summry nd onlusion, in ptients with ute pnretitis, we found signifint depletion of irulting lymphoytes whih ws muh more profound in the severe form of the disese. In the erly period of pnretitis, the mgnitude of peripherl depletion ws similr for T- nd B- lymphoytes. However, in the lte ourse of, B-lymphoytes were prtiulrly depleted. The seond week of the disese seems to be ritil for the ellulr immunity funtion, beuse of the strong shift in the CD7+/CD19+ rtio impliting predominne of T- over the B-lymphoytes in. Among T-lymphoytes, the signifint depletion of CD4+ popultion ws observed in nd, while CD8+ ells were in the norml rnge. It reted temporry imblne in the ell rtio with the pprent prevlene of CD8+ over the CD4+ ells. Serum IL-2, IFN-γ, TNF-α, IL-4, IL-5 nd IL-1 levels were signifintly inresed in both forms of AP, with the highest vlues found in. The mgnitude of elevtion of ytokines known to be produed by Th2 ells ws muh higher thn ytokines produed by Th1 ells. This finding suggests tht in the ourse of AP, Th1 subpopultion of CD4+ ells is suppressed more strongly thn Th2. The pttern of lymphoyte tivtion we found in AP ptients my be the onsequene of shift from Th1 to Th2. Strong elevtion of serum level of different ytokines, despite signifint derese in the number of irulting lymphoytes, my be explined by the signifint tivtion of T- s well s B-lymphoytes. Peripherl blood

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