Serum levels of IL-6 type cytokines and soluble IL-6 receptors in active B-cell chronic lymphocytic leukemia and in cladribine induced remission.

We have investigated the serum concentrations of interleukin-6 (IL-6) and two IL-6 family cytokines-oncostatin M (OSM) and leukemia inhibitory factor (LIF)-in 63 patients with B-cell chronic lymphocytic leukemia (B-CLL) and 17 healthy controls using the enzyme-linked immunosorbent assay (ELISA) method. Simultaneously, we measured the serum levels of the soluble forms of two subunits of the IL-6 receptor complex-ligand binding glycoprotein 80 (sIL-6R) and glycoprotein 130 (sgp130). The cytokines and receptors were evaluated in 25 untreated patients and 38 patients treated with cladribine (2-CdA), as well as in 17 healthy controls. We have correlated the serum levels of these proteins with Rai's clinical stage of the disease, the response to 2-CdA treatment and some hematological parameters. We have also evaluated the correlation of the IL-6 serum level with the concentration of OSM and IL-6 soluble receptors. IL-6 was measurable in 62/63 (98.4%), OSM in 20/25 (80%) of untreated and 14/38 (37.8%) of the treated patients. sIL-6R and sgp130 were detectable in all 63 patients and LIF in none of the CLL patients. IL-6 serum level in untreated patients was not significantly different as compared to its concentration in the control group (P>0.05). However, in the patients treated with 2-CdA the IL-6 level was significantly lower (P<0.02), and the lowest concentration was found in the patients with complete remission (CR; median 1.4pg/ml; P<0.02). The concentration of sIL-6R was significantly higher in untreated (median 61.8 ng/ml) and treated (median 50.1 ng/ml) CLL patients when compared to normal persons (median 41.2 ng/ml; P=0.04; P<0.001, respectively). There was no difference between the sIL-6R levels in the patients with CR and the healthy controls. In non-responders sIL-6R concentration was the highest and similar to its level in the untreated patients. OSM level was higher in the untreated patients (median 1.8pg/ml) than in the normal controls (median 0.0pg/ml; P<0.001) and in the CR patients (median 0.0pg/ml; P<0.03). The serum concentration of sgp130 was similar in the untreated (median 480 pg/ml) and treated (median 470 pg/ml) patients, as well as in the healthy persons (median 420 pg/ml; P>0.05). We have found significant positive correlation between the levels of sIL-6R and the lymphocytes count in CLL patients (p=0.423; P<0.001). In addition, sIL-6R and OSM serum concentrations correlated also with CLL Rai stage. In conclusion, the serum level of IL-6, OSM and sIL-6R, but not LIF and sgp130, are useful indicators of CLL activity.


Introduction
B-cell chronic lymphocytic leukemia (CLL) is the most common le uke mia in the We ste rn w orld, charac te rize d by the clonal prolife ration and ac cumulation of B lymphocytes. 1,2 These leukemic lymphocytes are charac te rize d by the ex pre ssion of CD5 marker and low de nsity monoclonal membrane immunoglobulin (Ig).
Interle ukin 2 (IL-2) and its rec eptors have be en show n to play a c entral role in the me chanism controlling the grow th of ne oplastic B ce lls. 3 ,4 Howe ve r, leukemic c ells in this disorder have also be en observe d to ex pre ss several other c ytokine rece ptors, including tumor nec rosis factors (TNF), 5 c olony stimulating factors (CSF) 3 and interle ukin-10 rec eptors. 6 On the othe r hand, the le ukemic cells in B-CLL can themselve s ex press and sec rete some cytokines including p roinflammatory cytokines, such as TNFa 7 ,8 and inte rleukin-6 (IL-6). 7,9 IL-6 is of spec ial inte re st in B-CLL, bec ause this cytokine acts as a B-c ell stimulatory factor (BSF-II), mediates B-ce ll diffe re ntiation and can stimulate the grow th of B-c ell lymphoid malignanc ie s such as myeloma. 10 In contrast, it has bee n proven that IL-6 is also able to inhibit TNF-a induce d prolife ration of B-cells from CLL patients. 1 1,1 2 IL-6 is a member of a family of cytokines w hic h also include s le ukemia inhibitory fac tor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and inte rleukin 11 (IL-11). 1 3,1 4 These cytokines are a group of evolutionary re late d prote ins characterize d by a c ommon tertiary framew ork w ith a distinctive four helix bundle topology. 1 5,1 6 IL-6 related cytokines have also a gp130 rec eptor compone nt involve d in the signal transduc tion across the c ell me mbrane , w hich ex plains the func tional pleiotropy and re dundancy of IL-6 type c ytokine s. 16 A consequenc e c ould be the involvement of these four cytokines, in a similar w ay to IL-6, in nume rous diseases including canc er, infe ctions and autoimmune diseases. 13 The ce llular IL-6 re ce ptor complex c onsists of tw o different prote ins [an 80-kDa ligand binding glyc oprote in (IL-6R) and gp 130] and is involved in c ellular signal transduc tion. 17 ,18 These tw o subunits of the IL-6R c omplex are proteolytically cleave d and release d from the c ell as soluble rec eptor prote ins. 1 9,2 0 Soluble forms of the IL-6R (sIL-6R) and gp130 (sgp130) have bee n found in diffe re nt body fluids in patients w ith various inflammatory and neoplastic disease s. 21 ,2 2 In the prese nt study w e have measured the serum conc entration of IL-6, LIF, OSM, as w e ll as sIL-6R and sgp130, in patients w ith active B-CLL and in patie nts w ith cladribine (2-chlorodeox yadenosine; 2-CdA) induc ed re mission. We have c orre late d the serum levels of the se proteins w ith the clinic al stage of disease according to Rai's staging syste m, and some he matological paramete rs. We have also e valuate d the correlation be tw ee n the serum le vels of IL-6 w ith both OSM and IL-6 soluble rec eptors.

Patients
The study c omprised 63 patients (31 fe male s and 32 males). The ir mean age w as 62.4 ye ars (range 41-81 ye ars). The characte ristics of the patie nts are show n Mediators of Inflammation · Vol 8 · 1999  2 3 At diagnosis the y had pe ripheral lymphocytosis greate r than 103 10 9 /l and more than 30% lymphoc ytes in normal to hyperce llular bone marrow. Immunologic ally all the p atie nts w ere CD5, CD19, CD20 and CD23 p ositive and show ed monoclonality for light chain immunoglobulin me mbrane surfac e rec eptors. Tw enty-five patie nts w e re previously untreate d and 38 had bee n pre viously treated w ith 2-CdA, w ith or w ithout prednisone . The treatme nt me thod has been described elsew here . 24 How ever, none of the p atie nts had be en treate d for at least 4 w eeks before the measureme nt of c ytokines. The clinic al stage of the dise ase ac cording to the Rai's classific ation 2 5 w as de te rmined at the time of the blood sample collection for c ytokine de te rmination. Guidelines for response in the previously tre ate d patie nts w e re those developed by the NCI sponsored Working Group. 23 Complete response (CR) required the absenc e of symptoms and organome galy, and a normal c omplete blood cell count (absolute neutrophil c ount > 13 10 9 /l and bone marrow w ith le ss than 30% of lymphoc ytes for at le ast 2 months). Re sidual disease w as e valuated in CR patients by immunophenotyping of pe ripheral blood and bone marrow. We have used a simultane ous dual color staining flow cytome try. Blood w as c ollec te d in vac uum tube s w ith EDTA as anticoagulant. Bone marrow ce lls w e re aspirated from the dorsal iliac cre st and immediately put into he parinized tubes. 2 6 Flow c ytometry analysis w as p erformed by EPICS-XL (Coulter, Hialeah, FL, USA). A combination of is o-thioc yanate (FITC) conjugate d monoclonal antibodies w as use d. Re sidual dise ase w as dete rmined by the coex pre ssion of CD5 /CD19 and CD5 /CD20 on B lymphoc ytes in c onjunc tion w ith monoclonality of surface light-chain ex pression on CD5 positive B ce lls. The prese nce of more than 10% of the total lymphoc ytic population coex pressing CD19 /CD5 and CD20 /CD5 w ith monotypic light chain ex pre ssion w as c onside red as monotypic light chain ex pre ssion. 26 Serum sampling and cytokine determination Venous blood sample s w ere c ollec te d in p yroge n fre e tubes, allow ed to clot at -4°C for 1 h and centrifuged at 2000 g for 10 min. The se rum obtained w as divide d into aliquots and stored at -70°C until assaye d for IL-6, OSM, LIF, sIL-6R ang gp130. The se ra w e re randomly code d and the te sting w as c arried out w ithout the know ledge of the clinical status of the subject or their related laboratory data. The cytokine se rum c oncentration w as assayed by spe cific, comme rc ially available, enzyme linke d assay (ELISA) kits (Quantikine , R&D Syste ms Inc , USA) in acc ordanc e w ith the manufac turer's instruc tions and analyze d w ith an ELISA re ader at 492 mm. The proc edure has bee n de scribe d in details elsew he re . 27 ,29 The sensitivity of the assay for IL-6 w as 0.7 pg /ml; for LIF 2.0 pg / ml and for OSM 2.1 pg /ml. Se rum for SIL-6R conce ntration measureme nt w as diluted 40 time s and its level w as measured betw e en 7.8 and 500 p g /ml. The minimum dete ctable dose of gp130 w as less than 0.05 ng /ml.

Statistical analysis
The me an value s w ere c ompared in Mann -Whitne y U te st and Kruskal-Wallis te st. Statistic al analysis for the freque ncy of dete ctable c ytokine s w as pe rforme d using Chi-square d te st. Zero value s, indic ating undete ctable le ve ls, w ere included in all the analyses. The linear c orre lations be tw ee n se rum inte rleukin le vels, as c ompared w ith the lymphocyte numbe r, w e re e valuate d using the Spe rman rank-sum correlation coeffic ie nt and linear regre ssion w ith the least square s me thod. The c omp arison and correlation w ere c onside re d significant w hen P< 0.05.

Results
The re sults of the measureme nt of IL-6, OSM, LIF, sIL-6R and sgp130 in untre ated patie nts and patients tre ate d w ith 2-CdA are show n in Table 2 In our study the IL-6 serum le vel in CLL patients (median 2.3 pg /ml) w as signific antly low er than in the he althy controls (median 6.3 pg /ml; P< 0.02). The low est conce ntration of this c ytokine has be en found in CR (median 1.4 pg /ml; P< 0.002; Table 3). How ever, the level of IL-6 in the untreated patients w as not significantly differe nt as compared to its c onc entration in the control group.
The se rum OSM le ve l w as significantly higher in the CLL patients (median 0.45 pg /ml), w hen c ompare d w ith the normal individuals (median 0.0 pg /ml; P< 0.002). Moreove r, it w as at its highest le ve l in the untreate d CLL patients (median 1.8 pg /ml). In contrast, the re w as no significant differe nce betw e en OSM le ve ls in the patients w ith CR and the c ontrol group (P> 0.05).
The serum conce ntration of sIL-6R w as significantly higher in the untreated (me dian 61.8 ng /ml) and tre ate d (median 50.1 ng /ml) CLL patients w hen compared to normal pe rsons (me dian 41.2 ng /ml; P< 0.001 and P= 0.04, respe ctive ly). There w as no difference be tw een the serum sIL-6R level in the patients w ith CR and the healthy control group (P< 0.05). More over, in patie nts w ho did not re spond to 2-CdA treatme nt, the sIL-6R conce ntration w as higher than in the responding patients, and similar to its le ve l in the untre ated group (P> 0.05).
The serum c onc entration of sgp130 w as similar in the untreate d (median 480 pg /ml) and treate d (median 470 pg /ml) patie nts as w ell as in the healthy persons (median 420 pg /ml). No corre lation betw e en the le ve l of the se soluble rec eptor and the response to tre atment w as found.
Mediators of Inflammation · Vol 8 · 1999 Surfac e immunofe notyping by flow c ytometry using the dual color staining te chnique on the peripheral blood and /or bone marrow w as performe d in all seven patients w ith 2-CdA induc ed CR. Residual dise ase w as demonstrated in three out of these patie nts. The se rum levels of investigate d cytokines and soluble rec eptors w e re similar in the patients w ith and w ithout residual disease (data not show n).
In the group of active CLL patients w e have also analyzed the re lationship betw e en the se rum conc entration of evaluate d cytokines, as w ell as the soluble rec eptors and the clinic al stage acc ording to Rai ( Table 4). The low est IL-6 c once ntration w as found in the patients w ith Rai 1&2 (median 1.5 pg /ml) as w e ll as those w ith Rai 0 (median 2.3 pg /ml); and the highest conc entration in Rai 3&4 patie nts (median 6.05 pg /ml).

Mediators of Inflammation · Vol 8 · 1999
281    The OSM se rum level w as highe r in the patients w ith Rai 0 and w ith Rai 1&2, w he n c ompared to the normal c ontrols (P< 0.001 and P< 0.03, respe ctively). Although the serum conc entration of this cytokine in the patients w ith Rai 3&4 w as not signific antly higher, w e observed that it w as detectable more ofte n in this group, w he n compare d w ith the controls.
How ever, the sIL-6R conc entration w as highe st in the patients w ith advanc ed Rai stage 1&2 (median 62.06 ng /ml) and 3&4 (me dian 53.26 ng /ml) and low est in patie nts w ith Rai O (me dian 46.69 ng /ml).
No differe nce betw e en the gp130 le ve l and clinic al stage of the patients w as obse rved.
The calc ulation of the sIL-6R to IL-6 ratio, as w ell as sgp130 to IL-6 ratio in the CLL patie nts and the he althy c ontrols, are prese nte d in Table 5. The sIL-6R/IL-6 ratio w as signific antly highe r in the treate d patients (me dian 23040.0) w hen c omp ared to untre ate d (median 22530.6) CLL patients (P< 0.0006). Moreover, it w as also higher in both groups than in the normal individuals (median 8209.7; P< 0.0006 and P< 0.02, re spe ctively). The highest ratio w as observe d in patients w ho did not respond to 2-CdA treatme nt (median 26510.0).
The re lationship betw e en the se rum c onc entration of e valuated c ytokine s as w ell as the soluble rece ptors and lymphocyte s c ount are show n in Fig. 1. We have only found a significant positive corre lation betw e en the le vels of sIL-6R and lymp hocyte s c ount in CLL patients (=0.423; P< 0.001). We have also analyzed the relationship be tw een serum levels of IL-6 w ith both OSM and the soluble rece ptors (Fig. 2). The only significant correlation w as observe d be tw een IL-6 and sIL-6 le vels in CLL patie nts (r = 0.244; P< 0.005).

Discussion
The role of c ytokines in the pathogene sis and the clinical course and the monitoring of ne oplasmatic diseases has bee n intensive ly investigate d for the last se veral years. It se ems that the most important cytokines, w hich play a ke y role in the pathogene sis of CLL, are IL-2, IL-4 and TNFa . 29 -32 The re sults of some studies also indic ate that IL-6 may play a role in the pathomechanism of CLL. 9,12 Other cytokine s belonging to the IL-6 family have not be en yet investigate d in CLL. In our study the c oncentration of three cytokines belonging to IL-6 family has be en me asured in the serum of 63 patients w ith B-CLL w ho had be en pre viously untre ated or w ho had re ceive d 2-CdA as a first line tre atment, including se ven patients w ho achie ve d a CR, and in a c ontrol group of 17 he althy voluntee rs. IL-6 w as measurable in all but one of the B-CLL patie nts and in all the healthy volunte ers. OSM w as dete ctable in 80% of untreated and in 37.8% of 2-CdA treated B-CLL patie nts, but w as me asurable only in 5.9% of the c ontrols. LIF w as undete ctable in all B-CLL patients, as w ell as in the control group.
The me an c onc entration of IL-6 in the se rum of untreate d patients w ith CLL w as not significantly different, w he n compare d to he althy controls. It is w orth stressing that IL-6 c oncentration w as low er in the tre ated CLL patients than in the untreate d subgroup (P< 0.02). We have also show n that IL-6 w as at its low est conce ntration in p atie nts in a CR upon 2-CdA (median 1.4 pg /ml), median in a PR (median 2.65 pg /ml), and at its highe st le ve l in non-responding patients (median 2.8 pg /ml). The results of other studies on IL-6 in CLL have re porte d e arlie r generally agre ed w ith our obse rvations. 33 ,3 4 Calle a e t a l. 3 4 show ed that the serum c onc entration of IL-6 is statistically highe r in patie nts w ith advanc ed or progressive stage of dise ase in comparison w ith smoulde ring B-CLL. Neverthele ss, there w as no statistic ally significant diffe renc e in IL-6 c onc entration betw e en B-CLL patie nts and he althy controls. Howe ve r, Auguilar-Sante lises e t a l. 33 show e d that in patients w ith monoclonal lymphocytosis of undete rmined significance (MLUS) IL-6 c oncentration w as tw ice as high as in healthy controls; but in B-CLL patients w ith stable as w ell as w ith in p rogre ssive disease the c oncentration of this cytokine w as similar to the control group.
The influe nce of treatme nt of c ytokine s levels in CLL has not be en, as yet, intensive ly studie d. Our observations indicate that IL-6 le ve l w as highe r in the patients w ith more advanc ed Rai stage s and at its low est in patients w ith 2-CdA induc ed remission. It is generally know n that 2-CdA ex erts a strong lymphocytolytic e ffec t against leuke mic B lymphocyte s, w hich is not se lective . This agent causes a long-lasting suppression of T CD4+ lymphoc yte s and monocytes w hich p roduce IL-6, in a similar w ay to leukemic B lymphoc ytes. 35 -37 It is supposed that the de crease of this cytokine in the se rum of CLL p atie nts upon 2-CdA tre atment, p artic ularly in those achie ving a CR, e ven below normal limits, may be ex plained by the 2-CdAinduc ed cytolytic e ffec t on various ce lls produc ing IL-6, including normal and leukemic lymphocytes. Dysregulation of the sec retion of IL-6 and other cytokines w as reporte d by Jew e ll e t a l. 38 in B-CLL patients treate d w ith INF-a .
Until now, LIF and OSM have not bee n w idely investigate d in patients w ith CLL. Lore got e t a l. 3 9 me asured LIF c oncentration in the serum of patie nts w ith various lymphoid malignanc ie s and show ed that its conce ntration w as increase d in Hodgkin's lymphoma (HL) and in non-Hodgkin's lymphoma (NHL), but in CLL and in healthy controls it remained low. In our study LIF conce ntration, measured w ith c ommercially available kits utilizing the ELISA method, w as undete ctable in CLL patients and in he althy c ontrols. It should be emphasized that similar re sults w ere earlier obtained in multiple mye loma, 28 syste mic lupus erythematode s 2 7 and rheumatoid arthritis. 22 In contrast, OSM w as detec table in a majority (80%) of untreate d patients w ith CLL and in only 37.8% of 2-CdA-tre ate d patients, as w ell as in only one (5.9%) he althy c ontrol. More over, the conce ntration of this cytokine w as signific antly higher in untre ated patients (median 1.8 pg /ml) than in 2-CdA treate d patients (median 0.0 pg /ml, P< 0.05), or than in healthy subjects (median 0.0; P< 0.001). The low est value s w ere note d in patients achie ving a CR. Similar re sults w ere re porte d e arlie r in multiple mye loma, w he re the OSM c onc entration w as statistically higher in p rogre ssive dise ase than in the c ontrol group. 2 8 The se observations are c oncomitant w ith those made by Koskela e t a l. 40 w ho dete cte d OSM in 20% of patie nts w ith multip le mye loma. Our results indic ate that OSM, like IL-6, may have a signific anc e in the pathogenesis of CLL and may be useful for the monitoring of the clinic al course and effective ness of therapy in lymphoid malignancie s.
Soluble IL-6 rec eptors (sIL-6R and sgp 130) are dete ctable in the serum and othe r body fluids in he althy c ontrols and in various pathological conditions. 1 7,20,2 2 sIL-6R has the agonist propertie s be cause it is able to bind IL-6 w ith an affinity almost like that of membrane IL-6R. Moreover, Il-6 /sIL-6R c omp lex is able to bind and activate the gp130 transducer chain. In our study the highest sIL-6R level w as observe d in the se rum of untre ated CLL patients, and the low e st values w ere note d in healthy controls and in CLL patients w ho e nte re d a CR (Table 3). Lavabre-Be rtrand e t a l. 2 0 also reported a signific antly higher sIL-6R conc entration in the serum of patients suffering from various lymphoid malignanc ie s, including CLL. The y show ed that the sIL-6R conce ntration w as highe r in more advanc ed stages of disease and sugge ste d its correlation w ith tumor ce ll mass. How e ve r, in contrast to our observations, the y did not show the influenc e of the rap y on the conce ntration of this soluble re ceptor. This disc repancy may be ex plaine d by the fact that their patients w ere tre ate d w ith the chlorambucil or CHOP sche dule and did not rec eive purine analogs; w hich may ex ert a more selec tive effect on the lymphoretic ular system, resulting in more c omplex re lationship betw e en the cytokines and the ir soluble re ce ptors. It is also know n that sIL-6R may be generated by the she dding of membrane IL-6R, as w e ll as by the translation of an alternatively splic ed RNA. 41 ,42 The signific ant positive corre lation betw e en the sIL-6R levels and the number of lymphocytes confirmes the observation made by Lavabre-Be rtrand e t a l. 2 0 that le ukemic B lymphocyte s are release d sIL-6R mainly via shedding. gp130 p lays a ke y role in signal transmission for all the cytokines of IL-6 family (Pete rs e t a l. 1 8 ). gp130 mRNA is ubiquitously ex pressed in almost all the ex amined organs, including the splee n, liver, lung, brain and he art. 4 3 In contrast to sIL-6R, the soluble form of gp130 show s antagonist p rope rties. Plasma sgp130 has be en show n to bind circulating IL-6 /sIL-6R complex and in this w ay to prevent ce lls ex pre ssing me mbrane gp130 from being activate d. 1 7 To our know le dge sgp 130 conc entration in the serum of patients w ith lymphoid neoplasmas has not be en investigate d as ye t. The c onc entration of sgp 130 w as similar in the control group and in the CLL patients, as w ell as in untre ated and 2-CdA treated p atie nts. The results of our studies suggest that dysregulation of this prote c tive me chanism may be involve d in the pathogenesis of CLL. We c an also state that this rec eptor plays a minor role as an indicator of the tumor mass or a marke r of the disease advance ment and therape utic effic ac y than sIL-6R, because its conc entrations in the se rum of CLL patients be fore and after tre atment and in healthy controls do not diffe r statistic ally. The differences in obse rvations conc erning these tw o soluble rec eptors are diffic ult to interp re t and the quantitative analysis of the de nsity of the se rece ptors on the membrane of le ukemic c ells may p rovide important information for the be tte r unde rstanding of the mechanism of the ir prote olytic cleavage in the soluble form.
In c onc lusion, w e have show e d that se rum conc entration of IL-6 is dec re ased, but OSM and sIL-6R are inc re ased in B-CLL patients in comparison w ith he althy controls and corre late s w ith the tumor mass and clinical stage of dise ase. More over, in patie nts achie ving a CR upon 2-CdA the le ve l of the se cytokines is similar to that in the control group, and in case of IL-6 it is even low er. The se rum c onc entration of sgp130 does not diffe r significantly betw e en CLL patients and he althy subjec ts, so its measurement has no practic al value in the monitoring of the dise ase activity and the efficacy of therapy.