In vitro studies on cellular and humoral chemotaxis in Crohn's disease using the under agarose gel technique*

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1 Gut, 1981, 22, In vitro studies on cellulr nd humorl chemotxis in Crohn's disese using the under grose gel technique* R D'AMELIO, P ROSSI, S LE MOLI, R RICCI, S MONTANO, AND F PALLONEt From the Deprtments of Medicine 3rd, Peditrics Ist, Clinicl Immunology nd Gstroenterology, University of Rome, Rome, Itly SUMMARY The locomotor function of polymorphonucler cells (cellulr chemotxis) nd serum chemotctic ctivity (humorl chemotxis) were studied in 51 ptients with Crohn's disese using method of migrtion under grose gel. To study cellulr chemotxis ptient's polymorphonucler cells were chllenged ginst norml Zymosn ctivted serum nd humorl chemotxis ws evluted testing the ptient's Zymosn ctivted serum ginst norml polymorphonucler cells. Cellulr chemotxis in the Crohn's disese group ws norml (lthough 3% of the 51 ptients hd migrtion vlues out of the norml rnge), while humorl chemotxis ws significntly lower in Crohn's disese ptients thn in the control group. However, the vlue of humorl chemotxis in the group of Crohn's disese ptients treted with steroids ws lower thn tht of ptients not treted, thus ccounting for the low men vlue observed in the Crohn's disese group s whole. The present results suggest tht defective chemotctic response my occur in some Crohn's disese ptients, prticulrly during steroid tretment. These findings might be relted either to defective genertion of complement derived chemotctic fctors or to the presence of circulting inhibitors. Leucocyte chemotxis is one of the erly stges of the phgocytic process which is the bsic mechnism in the clernce of prticulte or ntigenic mterils. In Crohn's disese defective clernce hs been suggested s one of the underlying pthogenetic mechnisms of the chronic inflmmtion.' Furthermore, the high prevlence of bscesses nd fistule in this condition suggests tht defects in locl defence mechnisms my occur during the nturl history of the disese. In studies using the skin window technique, Segl nd Loewi2 reported in Crohn's disese defective neutrophil function 'in vivo.' They found, however, norml neutrophil chemotxis 'in vitro' by modified Boyden chmber technique. Lter, Rhodes nd Jewell3 confirmed norml chemotxis 'in vitro' using different method of migrtion through millipore filters. *Supported in prt by grnt CNR no / to FP. taddress for correspondence nd reprints requests: F Pllone, Gstroenterology Unit, 2 Clinic Medic, Universit, vile del Policlinico, 161 Rom, Itly. Received for publiction 2 Jnury In the present investigtion the locomotor function of polymorphonucler cells (cellulr chemotxis) s well s the complement derived serum chemotctic ctivity (humorl chemotxis) were studied in Crohn's disese ptients using new method4 of migrtion under grose gel. As we hve lredy described4` defective chemotctic function in ptients with hypogmmglobulinemi nd/or IgA deficiency, the serum immunoglobulin levels were lso considered in the study. Methods PATIENTS Fifty-one ptients with Crohn's disese, 3 mle nd 21 femle, with men ge of 36.3 yers, were included in the study. Dignosis ws bsed on typicl clinicl nd rdiologicl findings nd confirmed by histology in 28 ptients. The men durtion of symptoms ws five yers. The disese involved the ileum (either s primry or recurrent disese) in 33 ptients, the ileum nd colon in 13, nd the colon only in five. Crohn's Disese Activity Index (CDAI) s proposed by the Ntionl Coopertive Crohn's 566 Gut: first published s /gut on 1 July Downloded from on 3 October 218 by guest. Protected by copyright.

2 PMN chemotxis in Crohn's disese Disese Study (NCCDS), USA6 ws used to evlute disese ctivity. Medicl tretment, when given, consisted of corticosteroids nd/or sulphslzine (Slzopyrin (SAP)). Twenty-nine ptients were receiving corticosteroids t the time of the study: men dose 17 mg/dy prednisone (rnge 5-25 mg) for men period of 5.5 months (rnge three to 1). Nine of these ptients were lso receiving SAP. Four ptients were receiving only SAP t the time of the study. Locl complictions were present in 16 ptients: enterocutneous fistule, eight; enteroenteric fistule, three; obstruction, two; nl lesions, three; bscesses, two. Two ptients hd systemic complictions: nkylosing spondylitis, one, nd pyoderm gngrenosum, one. CONTROLS Fifty-one helthy volunteers of comprble ge nd sex distribution were studied s control group. An dditionl 12 ptients with different chronic diseses (ulcertive colitis, five; chronic ctive heptitis, two; systemic lupus erythemtosus, three; sthm, two) chroniclly treted with steroids were lso considered. The results of liver function tests were within the norml rnges in ll ptients nd control subjects. Cell isoltion Smples of 1 ml heprinised sterile blood were centrifuged t 2 g for 15 minutes in polystyrene test tubes. The plsm nd buffy cot leucocytes were trnsferred to nother tube nd seprted by grvity sedimenttion t 37 C in mixture of ir (95%) nd CO2 (5%) for 45 minutes. The leucocyte-rich superntnt ws then lyered on Ficoll-Isopque density grdient nd centrifuged t 9 g for 3 minutes. The PMN were collected from the bottom nd resuspended in 3 ml of medium (TC 199, RPMI 164, Flow Lbortories). The cells were wshed twice nd the erythrocyte contmintion ws eliminted by lysis with buffered mmonium chloride. The cells were finlly resuspended t concentrtion of 3 x 16/ml. The medium used for resuspension ws the sme s tht used for preprtion ofgrose pltes. Collection ofserum nd ctivtion Both plsm EDTA nd fresh serum smples were collected. For ctivtion the fresh serum ws incubted for 3 minutes t 37 C with Zymosn (Sigm Chemicl Co) 5 mg/ml. It ws then centrifuged t 7 g for 1 minutes nd the superntnt collected. Preprtion ofgrose pltes Agrose (Reo 15, Boehring) ws dissolved in TC 199 or RPMI 164 by heting in boiling wter bth t concentrtion of 1.2%. After cooling to 45-5 C, the grose medium ws supplemented with 1% of fetl clf serum (Colordo serum Co) nd 1.5 of this solution ws poured into 35 mm tissue culture dish (31, Flcon) nd llowed to hrden. Three wells (4 mm in dimeter nd spced 2 mm prt) were cut, using suitble templte punch prepred by us. The grose plugs were removed by mens of Psteur pipette ttched to vcuum line. Leucocyte chemotxis ssy The tests were crried out in duplicte: 1,ul of the cell suspension ws dded to the centrl well nd 1,ul of ctivted serum to one of the lterl wells. The other wells ws filled with 1,ul of TC 199 or RPMI 164. The dishes were covered nd plced in 37 C humid tmosphere with 5% of CO2 for three hours. The preprtions were then fixed with My-Grunwld for 3 minutes, the lyer of the grose ws removed, nd the pltes stined with Giems. The migrted cells were counted in Leitz Orthopln microscope using x 1 eye-piece grid (side 1 cm) with x 1 mgnifiction. The number of cells which hd migrted towrds the non-ttrcting pole, which re considered to hve undergone rndom locomotion, ws subtrcted. After the preprtion hd been projected with Leitz microprojector (mgnifiction scle 1:175) the mximum distnce of migrtion ws mesured nd the distnce of rndom locomotion ws subtrcted. The mesurement ws then multiplied by the number of cells nd divided by 1. The Migrtion Index (MI) ws then clculted by the following formul: (no. chem. cells - no. rm cells) x (chem. d - rmd) 1 =MI The rndom migrtion index (RMI) ws clculted s: number rm cells x rmd 1 =RMI 567 where: chem.d= mximum migrtion distnce; rmd distnce of rndom migrtion; chem. cells=pmn chemotxis migrted; rm cells=pmn rndomly migrting. Using this leucocyte chemotxis ssy we evluted the cellulr chemotctic function by testing the ptients PMN ginst norml Zymosn ctivted serum (ZAS). To study the humorl chemotxistht is, the genertion of complement derived serum chemotctic fctors-norml PMN were chllenged ginst ptient's ZAS. Complement nd immunoglobulin ssy Plsm EDTA C3c nd C4 levels nd serum immunoglobulins A, G, M were detected by the method of Gut: first published s /gut on 1 July Downloded from on 3 October 218 by guest. Protected by copyright.

3 568 Tble D'Amelio, Rossi, Le Moli, Ricci, Montno, nd Pllone Vlues (men+sd) of migrtion index for cellulr nd humorl chemotxis in control subjects nd Crohn's disese Control Crohn's disese Ptients Treted Untreted Cellulr chemotxis NS 477± ±426 NS 47±45 Humorl chemotxis 492±25 P< ( 372± P<.2 458±225 Mncini et l. The C3, ws evluted using specific ntiserum (Boehring-werke). The immunoglobulin nd complement ssys were performed in 48 ptients. For the sttisticl nlysis of the dt Student's t test ws used. Results No significnt difference ws found between helthy controls nd Crohn's disese ptients s fr s the men vlues of polymorphonucler cellulr chemotxis re concerned (Tble 1). However nine out of the 51 Crohn's disese ptients hd migrtion index below the lower norml limit, while, in seven ptients, migrtion index bove the upper norml limit ws observed (Fig. 1). No correltion ws found between cellulr chemotxis vlues nd site nd extent of x 'm.2 7 E * 3 2..e *. 2 bowel involvement, disese ctivity, nd occurrence of complictions. Seven out of nine Crohn's disese ptients with low vlues were receiving steroid tretment t the time of the study. No low vlues were found in the steroid treted control group. The men vlues of humorl chemotxis-tht is, complement derived serum chemotctic ctivitywere significntly lower in Crohn's disese ptients thn in the helthy controls (Tble). Agin, no significnt correltion ws found between migrtion index nd disese ctivity, occurrence of complictions, nd site of bowel involvement. However when the humorl chemotxis vlues were correlted with the medicl tretment (Fig. 2) significntly lower men vlue of migrtion index ws observed in the steroid treted Crohn's disese ptients. The men vlues of IgG, IgA, nd IgM were not significntly different from those of the norml 1. * I 1 A 1 gog go * * go x c nrsn ulwj c 7 [I--LFAzz.xL4... I ] t b c d e b c d e Fig. 2 Vlues ofpolymorphonucler humorl chemotxis Fig. 1 Vlues ofpolymorphonucler cellulr chemotxis expressed s Migrtion Index (MI). Horizontl brs indicte expressed s Migrtion Index (MI). Horizontl brs indicte men vlues... norml limits (men±2sd). : helthy men vlues... norml limits (men+2sd). : helthy controls; b: whole Crohn's disese group; c: steroid treted controls; b: whole Crohn's disese group; c: steroid treted Crohn's disese ptients; d: untreted Crohn's disese Crohn's disese ptients; d: untreted Crohn's disese ptients; e: steroid treted controls. P<1, b vs ; P<2, d ptients; e: steroid treted controls. vs c r _ _... ;3. * * t **i' 3 _,@, :. S Gut: first published s /gut on 1 July Downloded from on 3 October 218 by guest. Protected by copyright.

4 PMN chemotxis in Crohn's disese E l- 12' 6.* *: &. 4 1.Is( _ U. :; * l^m _ mow * *:,* *I. f*: JLL IgG 1gA lgm U& 1. * Sm.2L * - "W m * * OUm 5 controls. No significnt difference ws found between steroid treted nd non-treted ptients, s fr s the serum Ig levels were concerned (Fig. 3). Men vlues of plsm EDTA C3, were norml, lthough nine out of 48 ptients hd C3, levels bove the upper norml limit. C4 levels were within the norml rnge in ll ptients (Fig. 3). Discussion - j 11 1 The results of this study show tht in Crohn's disese ptients men vlues of polymorphonucler cellulr chemotxis were norml (lthough bout 3% vlues were not within the norml rnge), while humorl chemotxis vlues-tht is, complement derived serum chemotxis ctivity-were significntly lower thn those of helthy controls. In fct, impired humorl chemotxis occurred mostly in the steroid treted ptients, thus ccounting for the low men vlue observed in the Crohn's disese group s whole. The men vlue nd distribution of the group of Crohn's disese ptients not receiving steroids were not different from those of the helthy control group. In the steroid treted control group no decrese in humorl nd cellulr chemotxis ws observed. Controversil dt hve been reported on the effects of corticosteroids on chemotxis in vitro. Wrd7-9, using the Boyden chmber technique, showed tht the ddition of hydrocortisone nd/or methylprednisolone in vitro suppressed the polymorphonuclerchemotctic response to complementderived chemotctic fctors. This effect ppered to C3 *8S U.to C4 Fig. 3 Serum immunoglobulins nd C3 nd C, levels in 49 Crohn's disese ptients. untreted ptients. E steroid treted ptients norml... limits. 569 be irreversible nd the drugs ppered to ct directly on polymorphonucler cells stbilising lysosoml membrnes.7 Other reports showed no effect of corticosteroids on in vitro chemotxis, in humns1 1 nd in rbbit.`2 Recently Clrk etl. 13 showed tht the dministrtion of prednisone to helthy people hd no effect on their polymorphonucler cells chemotxis tested in vitro. In the present investigtion no difference ws found between Crohn's disese ptients treted with steroids nd those not treted s fr s the cellulr chemotxis ws concerned, but n impired serum chemotctic ctivity ws observed in those receiving corticosteroids. A defective genertion or n incresed consumption of complement derived chemotctic fctors could be dvnced s mechnism for these findings. In fct, there is evidence`4 demonstrting tht C' 1 ctivtion does occur in Crohn's disese, lthough serum C3 levels re norml or incresed. Therefore, n incresed consumption of complement derived chemotctic fctors could contribute in decresing the serum chemotctic ctivity. Such n explntion does not ccount, however, for the significnt difference observed in the Crohn's disese group between treted nd untreted ptients. Chemotctic fctor inctivtors re described in norml subjects. 6 Their function is to control norml chemotctic response. Cell-directed humorl suppressor substnces re present in the serum of ptients with srcoidosis in higher concentrtion thn norml serum' 18 nd similr findings were reported in Hodgkin's disese'9 nd in lepromtous leprosy.2 Gut: first published s /gut on 1 July Downloded from on 3 October 218 by guest. Protected by copyright.

5 57 D'Amelio, Rossi, Le Moli, Ricci, Montno, nd Pllone It might, therefore, be speculted tht similr fctors re present in high concentrtion in the serum of Crohn's disese ptients, prticulrly in those receiving steroid tretment. In summry, the present results suggest tht defective complement derived serum chemotctic ctivity my occur in some ptients during steroid tretment. These findings my offer n explntion for the therpeutic effect of corticosteroids in Crohn's disese. On the other hnd, s lredy speculted by Segl nd Loewi,2 they suggest tht steroid tretment might ply role in promoting chronicity. Additionl studies re necessry on the possible reltionship between chemotctic function, medicl tretment, nd the nturl history of Crohn's disese. The uthors re much indebted to Professor F Aiuti, Professor Luis Businco, nd Professor A Torsoli for their helpful criticisms nd dvice. References 'Wrd M. The pthogenesis of Crohn's disese. Lncet 1977; 2: Segl AW, Loewi E. Neutrophil dysfunction in Crohn's disese. Lncet 1976; 2: Rhodes JM, Jewell DP. White cell chemotxis in Crohn's disese nd ulcertive colitis. Gut 1979; 2: A436. 4D'Amelio R, Le Moli S, Rossi P, Aiuti F. Neutrophil chemotxis defect in IgA deficiency evluted by migrtion grose method. Scnd J Immunol 198; 11: 'D'Amelio R, Rossi P, Le Moli S, Aiuti F. Defective neutrophil chemotxis in hypogmm-globulinemi nd selective IgA deficiency. Clin Immunol Immunopthol 198; 16: Best WR, Becktel JM, Singleton JW, Kern F. Development of Crohn's disese ctivity index. Gstroenterology 1976; 7: 'Wrd PA. The chemosuppression of chemotxis. J Exp Med 1966; 124: Wrd PA. Chemotxis of polymorphonucler leukocytes. Biochem Phnncol Suppl 1968; Wrd PA, Leukotctic fctors in helth nd disese. Am. J Pthol 1971; 64: '`Sorkin E, Stecher VJ, Borel JF. Chemotxis of leukocytes nd inflmmtion. Ser Hemtol 197; 3: "Losito A, Gwyn Willims D, Cooke G, Hrris L. The effect on polymorphonucler leucocyte function of prednisolone nd zthioprine nd 6- mercptopurine in vitro. Clin exp Immunol 1978; 32: '2Borel JF. Effect of some drugs on the chemotxis of rbbit neutrophils in vitro. Experimenti 1973; 29: '3Clrk RAF, Gllin JI, Fuci A. Effects of in vivo prednisone on in vitro eosinophil nd neutrophil dherence nd chemotxis. Blood 1979; 53: '4Hodgson HJF, Potter BJ, Jewell DP. Humorl immune system in inflmmtory bowel disese: I Complement levels. Gut 1977; 18: '5Hodgson HJF, Potter BJ, Jewell DP. C3 metbolism in ulcertive colitis nd Crohn's disese. Clin Exp Immunol 1977; 28: `Berenberg JL, Wrd PA. The chemotctic fctor inctivtor in norml humn serum. J Clin Invest 1973; 52: '7Kntor FS, Dwyer JM. Srcoid. J Invest Derm 1976; 67: '8Gnge RW, Blck MM, Crrington P, McKerron R. Defective neutrophil migrtion in srcoidosis. Lncet 1977; 2: '9Wrd PA, Berenberg JL. Defective regultion of inflmmtory meditors in Hodgkin's disese: supernorml levels of chemotctic fctor inctivtor. N Engl J Med 1974; 29: Wrd PA, Gorlnick S, Bullock WE. Defective leukotxis in ptients with lepromtous leprosy. J Lb Clin Med 1976; 87: Gut: first published s /gut on 1 July Downloded from on 3 October 218 by guest. Protected by copyright.