Soluble complement receptor 1 is increased in patients with leukemia and after administration of granulocyte colony-stimulating factor Salima Sadallah, Estelle Lach, Sybille Schwarz, Alois Gratwohl,* Olivier Spertini,† and Ju¨rg-Alfred Schifferli Immunonephrologie Labor and *Abteilung fu¨r Ha¨matologie, Department Forschung, Kantonsspital Basel; and †Division d’He ´ matologie, De´partement de Me´decine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
Abstract: Complement receptor type 1 is expressed by erythrocytes and most leukocytes. A soluble form is shed from the leukocytes and found in plasma (sCR1). sCR1 is a powerful inhibitor of complement. We report an increased sCR1 in the plasma of leukemia patients, up to levels producing measurable complement inhibition. Half of the 180 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) had sCR1 levels above the normal range. The highest levels were observed in T-ALL (17 patients). The complement function of a T-ALL serum was improved by blocking sCR1 with a specific mAb (3D9). Measurements in 16 peripheral stem cell donors before and after granulocyte colony-stimulating factor (G-CSF) administration showed an increase in sCR1 (before, 43.8 6 15.4; at day 5, 118.3 6 44.7 ng/mL; P F 0.0001). This increase paralleled the increase in total leukocyte counts and was concomitant with de novo leukocyte mRNA CR1 expression in all three individuals tested. Whether pharmacological intervention may be used to up-regulate sCR1 so as to inhibit complement in vivo should be further investigated. J. Leukoc. Biol. 65: 94–101; 1999. Key Words: plasma · monoclonal antibody 3D9 · stem cells
INTRODUCTION Complement receptor type 1 (CR1, CD35, C3b/C4b receptor) is an integral membrane glycoprotein expressed on most circulating cells, including erythrocytes, polymorphonuclear leukocytes, monocytes, B lymphocytes, and some T lymphocytes [1–3]. On phagocytes, CR1 is mainly involved in the initial binding of C3b-coated particles that are subsequently ingested [4]. In addition, CR1 serves as a cofactor for the cleavage by factor I of C3b to C3bi and C3dg [5]. These degradation products of C3b are the ligands for other receptors (CR3/CR4 and CR2, respectively) so that phagocytosis and immune responses are enhanced. The cleavage of C3b interrupts further complement activation and the formation of the terminal complement complex. Thus, CR1 is a potent cofactor for the 94
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inhibition of complement activation. Weisman et al. have estimated that the recombinant soluble form of CR1 is at least 100-fold more efficient for the inactivation of C3b than factor H, the physiological cofactor in plasma [6]. In vitro, leukocytes that express CR1 on their surface release a soluble form of CR1 (sCR1) by proteolytic cleavage [7]. The intracellular and transmembrane domains of CR1 are very small (estimated Mr < 6,200), whereas the extracellular domain is large (Mr < 204,000). The sCR1 fragment released by leukocytes has an estimated Mr 200,000–210,000 very similar to the whole CR1, indicating that sCR1 corresponds to most of the extracellular domain of CR1. A soluble form of CR1 is present in the plasma at a low concentration [8–10]. Soluble CR1 retains its functional activity in plasma, although its low concentration precludes any role in controlling complement activity. Soluble CR1 in plasma has the same size and is recognized by the same monoclonal antibody as its counterpart shed by leukocytes in vitro. Because CR1 is mostly expressed by hematopoietic cells, plasma sCR1 is likely to derive from these cells by continuous shedding. In a preliminary study on a small group of patients with hematologic malignancies, sCR1 levels were found to be increased in some patients and to drop at the time of chemotherapy [9]. L-selectin is an adhesion molecule that initiates leukocyte attachment to activated endothelium [11, 12]. Its primary structure exhibits similarities to CR1 because both molecules have an extracellular backbone of short consensus repeats as found in many complement regulatory proteins. In addition, L-selectin is shed from leukocytes by proteolytic cleavage and a shed form corresponding to a large segment of the extracellular domain is found in plasma (sL-selectin) [13]. Patients with acute leukemia have increased levels of sL-selectin that correlate with the disease severity [14]. This study was undertaken to define the frequency of sCR1 increase in leukemia, its correlation with sL-selectin, and to
Abbreviations: G-CSF, granulocyte colony-stimulating factor; sCR1, soluble complement receptor 1; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; ELISA, enzyme-linked immunosorbent assay. Correspondence: Dr. S. Sadallah, Immunonephrologie Labor, Department Forschung, Kantonsspital Basel, Hebelstrasse 20, 4031 Basel, Switzerland. E-mail:
[email protected] Received May 26, 1998; revised October 7, 1998; accepted October 8, 1998.
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evaluate potential biological effects of an increase in sCR1 on complement function. In addition, we wanted to examine the regulation of the levels of soluble receptors in vivo and analyzed plasma and cells of individuals who received G-CSF before peripheral stem cell donation.
vesicle-bound CR1 of erythrocyte-derived and podocyte-derived vesicles [16, 17] as well as leukocyte membranes [18]. The size of sCR1 in plasma was assessed by gel filtration on FPLC (Pharmacia). The total-CR1 ELISA was performed on the fractions eluted from two normal donors, a leukemia patient and a stem cell donor after G-CSF administration.
PATIENTS AND METHODS
Cell purification, RNA isolation, and Northern blot analysis
Study design This study was a retrospective analysis of samples stored in liquid nitrogen by the Swiss Working Group Leukemia of the Swiss Institute for Clinical Cancer Research (SIAK) and the hematology laboratory Kantonsspital Basel. Examination of samples from normal stem cell donors was part of a prospective study evaluating the effect of G-CSF on normal donors. The population was made up of 180 patients with newly diagnosed leukemia. Among them: 95 patients with acute myeloid leukemia (AML), 15 M1, 48 M2, 7 M3, 17 M4, and 8 M5; 75 patients with acute lymphoblastic leukemia (ALL), 17 had T-ALL and 58 had lymphoblasts expressing B-lineage markers; and 10 patients with chronic lymphocytic leukemia (CLL). The diagnosis and classification of AML, ALL, and CLL were based on the criteria of the French-AmericanBritish Cooperative Group [15] and immunophenotyping was performed as previously reported [14]. The same patients previously studied for sL-selectin have been included in this study population. Heparinized plasma samples had been stored either in liquid nitrogen or at 275°C. Plasma samples were obtained from 16 normal healthy individuals who served as peripheral stem cell donors for allogenic transplantation before and after G-CSF (10 µg/kg/day) administration for 5–12 days.
Measurements of sCR 1 and soluble L-selectin Soluble CR1 levels were determined in plasma with a sandwich enzyme-linked immunosorbent assay (ELISA), using two anti-CR1 monoclonal Ab (3D9 and J3D3), that recognizes two different epitopes on the extracellular portion of CR1 (total-CR1 ELISA) [9]. Recombinant soluble CR1 (rsCR1) was used as a standard (a kind gift from Dr. Ryan, T cell Sciences). The assay was performed in the presence of 0.05% Tween after centrifugation of the samples at 3,000 g for 10 min at 4°C. The limit of sensitivity was 0.02 ng/mL. Soluble L-selectin concentration was measured with a sandwich ELISA [12]. This simple, quantitative sandwich ELISA uses two monoclonal antibodies directed against the extracellular domain of L-selectin. The anti-LAM1-5 monoclonal antibody was used as a capture antibody. The presence of sL-selectin was revealed with biotinylated anti-LAM1-3 monoclonal antibody [14]. The assay has a detection range of 5–1,300 ng/mL, is precise, and is sensitive. The ability of this assay to detect sL-selectin in serum, plasma, and culture supernatant fluid was demonstrated and it was used to quantitate circulating sL-selectin in normal and patient sera [12, 14]. Plasma samples were diluted at 1/100 to 1/2,000 to obtain a concentration in the linear range of the assay (20–60 ng/mL). The sL-selectin level in the reference plasma was measured using a recombinant L-selectin as the standard. The chimeric protein was produced in COS-1 cells transfected with pL-selectin/g1 cDNA subcloned into the AprM8 expression vector provided by L. Klickstein (Center for Blood Research, Boston, MA) [14]. All assays were performed in duplicate or triplicate.
Analysis of sCR1 in plasma The ELISA specific for transmembrane CR1 (m-CR1) was performed as described previously [7]. In this sandwich assay the first Ab is a polyclonal rabbit IgG Ab recognizing a synthetic peptide corresponding to the last 19 amino acids of the intracytoplasmic tail of the CR1 molecule, and the second is a monoclonal Ab recognizing an epitope on the extracellular portion of CR1 (J3D3). Thus, only CR1 bearing the intracytoplasmic tail is recognized in this assay. This assay was performed in the presence of 0.5% Triton X-100 and standardized with a known amount of erythrocyte CR1. The limit of sensitivity was 0.1 ng/mL. The solubility of CR1 in plasma was assessed before and after ultracentrifugation at 200,000 g at 4°C for 1 h. This centrifugation has been shown to pellet
Blood from peripheral stem cell donors before and after G-CSF administration was obtained by venipuncture. Total leukocytes were isolated under sterile conditions by Ficoll-Hypaque density gradient centrifugation as described [19]. Total RNA was extracted from cell preparation by the guanidine-thiocyanate method [20] and electrophoresed on 1.1% agarose gels blotted onto Hybond N1 (Amersham). Fixation of the RNA was done by UV cross-linking. Hybridizations were performed overnight at 70°C with the use of a digoxigenin [21] (Dig)-labeled UTP cRNA probe specific for CR1. The CR1 mRNA-specific probe was kindly provided by W. W. Wong (Brigham and Women’s Hospital, Boston, MA) [22]. The hybridized digoxigenin-labeled probe was detected with an anti-Dig alkaline phosphatase-coupled antibody. The alkaline phosphatase reaction was performed with chemiluminescence substrate CSPD (protocol of Boehringer Mannheim). The reaction product was visualized by exposure to BioMaxMR films (Kodak). Methylene blue staining was used as control for the amounts of RNA loaded onto the gels [23].
Inhibition of complement function by sCR1 Inhibition of the complement-dependent lysis of sheep erythrocytes sensitized with rabbit IgM (EA). The EA (108 cells/mL) were incubated for 30 min at 37°C with human serum diluted 1/180 in the presence of increasing amounts of srCR1. The lysis was assessed by hemoglobin release in the supernatant. rsCR1 was added so as to increase the normal serum concentration of sCR1 by 6- and 15-fold. In a second series of experiments rsCR1 was added to increase CR1 by 6-fold followed by a 100-fold molar excess of a mAb anti-CR1 (3D9) known to inhibit CR1 function. The effect of the mAb alone served as a control. All results were expressed in percent inhibition of EA lysis. Experiments were done in duplicate. Inhibition of complement activation by human heat-aggregated IgG. Normal human IgG (10 mg/mL) was aggregated at 61°C for 1 h and large aggregates were removed by centrifugation at 10,000 g for 10 min. Normal human serum diluted 1/5 was incubated with a fixed amount of aggregated IgG (1 µg aggregated IgG/1 µL serum) for 20 min at 37°C and rsCR1 so as to increase the normal serum concentration of CR1 by 10- and 100-fold. The reactions were stopped by adding EDTA and the concentration of C5a des-Arg was measured using a commercial kit (Amersham). Normal serum was supplemented simultaneously with a 10-fold excess of rsCR1 and a 100-fold molar excess of anti-CR1 mAb (3D9) before being exposed to IgG aggregates. The effect of the mAb alone served as a control. The results were expressed in percent inhibition of C5a produced compared with the control experiment performed without addition of rsCR1. Experiments were done in duplicate. Inhibition of the hemolytic alternative pathway. Rabbit erythrocytes were incubated for 30 min at 37°C with human serum diluted 1/13 in the presence of 2 mM Mg and 10 mM EGTA [24]. rsCR1 was added to increase the normal serum concentration of CR1 to 10-fold. The reaction was stopped by adding 20 mM EDTA. After centrifugation the supernatant OD was read at 541 nm. The control experiment was done without addition of rsCR1. The experiment was repeated in the presence of a 100-fold molar excess mAb anti-CR1 (3D9). The T-ALL patient who had a 20-fold increase in serum CR1 was studied in this assay with and without adding a 100-fold molar excess anti-CR1 mAb (3D9). Experiments were done in triplicate.
Statistical analysis The Mann-Whitney U test has been used for all comparisons of sCR1 plasma concentrations. The correlations between sCR1 and sL-selectin; sCR1 and the total leukocyte counts (Spearman rank test Rs) were performed with the StatView SE1Graphicsy program. sCR1 and sL-selectin were measured in the same samples of 172 patients, a same-day leukocyte count was available for only 85 of the sCR1 measurements.
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RESULTS Soluble CR1 levels in plasma of patients with leukemia The mean level of sCR1 was 31.4 ng/mL (min, 17.8; max, 55.7 ng/mL) in 30 normal individuals, 53.5 ng/mL (min, 13; max, 123) in 95 patients with different forms of AML, 60.34 ng/mL (min, 16.5; max, 142.7) in the 58 patients with ALL (non -T-), 185.34 ng/mL (min, 16; max, 872) in the 17 T-ALL patients, and 49.3 ng/mL (min, 16; max, 103) in 10 patients with CLL (Fig. 1). There was a significant increase in sCR1 levels P , 0.001 in all the different groups of leukemia compared to the group of normal individuals but not in the CLL patient group P , 0.3. Fifty-six percent of the patients (101 out of 180) had a sCR1 level above the normal range (P , 0.001; i.e., 61% of 75 patients with ALL, 40% of 10 with CLL, and 47% of 95 with AML). In the ALL patients there was a significant difference in plasma sCR1 level between T-ALL patients (P , 0.002) and the 58 patients who had lymphoblasts expressing B-lineage markers, i.e., ALL (non -T-). Fourteen out of seventeen patients with T-ALL had levels above the normal range, and in five of these the sCR1 was equal or higher than 10-fold the normal mean. This was surprising because under normal circumstances T lymphocytes are known to express only a few or no CR1 [25–27]. No cells from these patients were available to determine whether the T-ALL cells expressed CR1.
Soluble CR1 with soluble L-selectin levels and total leukocyte count Soluble L-selectin is increased in patients with leukemia [14]. sL-selectin is thought to be shed by blast cells of both origins,
Fig. 2. Correlations between (A) sCR1 and sL-selectin levels and (B) sCR1 and leukocytes in ALL patients. The filled circles represent patients with T-ALL.
lymphoblastic and myeloblastic. In addition, the sL-selectin level correlates with the total mass of blast cells in the body and only weakly with peripheral venous blast cell counts [14]. We found a weak but significant correlation between sCR1 and sL-selectin (n 5 172; Rs 5 0.34, P , 0.001) in patients with leukemia. A subgroup analysis indicated that this correlation was due mainly to patients with ALL (n 5 75; Rs 5 0.428, P , 0.001; Fig. 2A). Soluble CR1 correlated strongly with venous leukocyte counts in patients with ALL (n 5 33; Rs 5 0.5, P , 0.006; Fig. 2B), not in the remainder.
Effect of G-CSF administration on sCR1 levels
Fig. 1. sCR1 levels in plasma of 30 normal blood donors, 95 patients with AML, 58 patients with ALL (non -T-), 17 patients with T-ALL, and 10 patients with CLL. Median values are indicated by horizontal bars. There was a significant increase in sCR1 levels P , 0.001 in all the different groups of leukemia compared to the group of normal individuals but not in the CLL patient group P , 0.3.
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Because it has been reported that growth factors up-regulate the surface expression of CR1 in leukocytes [28–34], we wanted to determine whether the administration of G-CSF would increase the levels of sCR1 in vivo. In normal individuals who were prepared for peripheral stem cell donation, the administration of 10 µg/kg of G-CSF produced a rapid rise in sCR1 that paralleled the increase in leukocyte count (Fig. 3). The increase was approximately 2.6-fold after 5 days (n 5 16, from 44.8 6 15.4 to 118.3 6 44.7 ng/mL; P , 0.001; Fig. 4A). Northern blot analysis of RNA purified from total leukocytes of three peripheral stem cell donors before and after G-CSF administration was done to investigate any modification in the CR1 mRNA expression. The RNA from the donors had the 9and 11-kb CR1 mRNA species previously described for PBMC mRNA [35]. CR1 mRNA was weakly detectable in the donors http://www.jleukbio.org
Fig. 5. Northern blot analysis of total leukocyte RNA from two peripheral stem cell donors. An increased expression in the 9-kb and 11-kb CR1 mRNA species was detected already 3 days after G-CSF administration. The 5-kb band on the Northern blot represents the size of the ribosomal RNA 28s.
Characteristics of sCR1 in leukemia Fig. 3. sCR1 levels and total leukocyte counts in a peripheral stem cell donor during G-CSF administration.
before G-CSF administration and the expression increased 3–5 days after administration (Fig. 5). The effect of G-CSF was not limited to sCR1 because sL-selectin increased as well from 1.54 6 0.49 µg/mL up to 3.58 6 0.96 (P , 0.001) at day 5, corresponding to a 2.3-fold increase, which was very similar to the sCR1 increase (Fig. 4B).
Fig. 4. Increase in (A) sCR1 and (B) sL-selectin levels in 15 peripheral stem cell donors before and after 5 days of G-CSF administration.
Several experiments were undertaken to determine whether the sCR1 in patients with leukemia was modified compared to sCR1 of normal individuals. None of the results obtained provided any significant difference. Ultracentrifugation is known to pellet CR1 bound to vesicles derived from erythrocytes and podocytes, as well as CR1 bound to polymorphonuclear leukocyte membranes [16–18]. By contrast, sCR1 is not pelleted by ultracentrifugation [8, 9]. There was a small loss of CR1 after ultracentrifugation of the plasma from leukemia patients with high levels of sCR1, a minimal loss after G-CSF administration, and no loss in a normal control (Fig. 6). Because the samples of the leukemia patients had been stored for prolonged periods of time, we considered this loss to be nonspecific. The CR1 attached to microvesicles released from cells corresponds to the whole transmembrane CR1 [18]. The ELISA specific for transmembrane CR1 (mCR1) [7] does not recognize sCR1 (see Patients and Methods). Only a small fraction (,7%) of CR1 measured in plasma corresponded to transmembrane CR1, indicating that the high levels observed were not explained by the presence of uncleaved CR1 (not shown). Finally, gel filtration on FPLC showed no difference in the size and no fragmentation of sCR1 when plasma from normal subjects, leukemia patients, and stem cell donors after G-CSF
Fig. 6. CR1 levels in plasma before and after ultracentrifugation at 200,000 g for 60 min in the following five individuals: 1, T-ALL; 2, AML (M2); 3 and 4, peripheral stem cell donors; 5, normal donor.
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administration were compared (not shown). In conclusion, plasma CR1 in leukemia and after G-CSF administration was not attached to vesicle, had lost its intracellular domain, was of similar size as sCR1 of normal plasma, and was not fragmented.
Effect of sCR1 in excess on complement function in vitro The concentration of sCR1 in normal plasma is too low (< 0.15 nM) to have an effect on complement function. However, the data obtained by Weisman et al. [6] do not exclude that a high level of sCR1 as observed in some of the patients with T-ALL may have some effects on complement activity. Thus, we studied complement activation in normal serum supplemented with recombinant sCR1. First, we tested the effect of a 6- and 15-fold increase in the sCR1 level on classical pathway activity using sheep erythrocytes sensitized with IgM. In the control experiments the lysis was 68 6 4% and the results were expressed in percentage inhibition of erythrocyte lysis. The increase in sCR1 produced a dose-dependent inhibition of classical pathway activity (Fig. 7). We then investigated whether this effect was specific with the use of a mAb (3D9) known to block CR1 function. The addition of a 100-fold molar excess of the mAb over CR1 reverted the inhibition produced by CR1 although the effect was not complete (Fig. 7). We tested the effect of rsCR1 on the production of C5a by heat-aggregated IgG in normal serum, an assay that requires both classical and alternative pathway function [6]. As shown in Figure 8, a 10-fold increase in CR1 produced a significant inhibition of C5a release, and this effect was reverted by anti-CR1 mAb. Finally we turned to a hemolytic assay for alternative pathway function using rabbit erythrocytes. In this assay a 10-fold increase in sCR1 significantly inhibited the alternative pathway activity. The addition of a 100-fold excess anti-CR1 mAb reverted the alternative pathway function to the initial level (Fig. 9). Adding mAb to normal serum did not change the hemolysis (not shown). This assay was used to test the serum of the T-ALL patient who had the highest level of sCR1. Because the differences were small, it was repeated three times in
Fig. 7. Percentage inhibition of the complement-dependent lysis of sheep erythrocytes (E) sensitized with rabbit IgM (EA). The inhibition of lysis due to rsCR1 was partially reverted by a monoclonal anti-CR1 Ab (3D9) added in a 100-fold molar excess.
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Fig. 8. Percentage inhibition of C5a-des-Arg generation induced by heataggregated IgG. The inhibition of C5a generation due to rsCR1 was reverted by the monoclonal anti-CR1 Ab (3D9) added in a 100-fold molar excess.
triplicate, and each time mAb anti-CR1 excess produced a significant (P , 0.003) improvement in complement function (Fig. 9 shows the mean results of the three experiments combined).
DISCUSSION In this study we show that sCR1, the cleaved and shed form of the transmembrane CR1, is present at high concentrations in plasma in more than half the patients with leukemia, at a level sufficient to down-regulate complement function. The possible mechanisms responsible for high levels of sCR1 in humans include increased synthesis by leukocytes induced by growth factors. The majority (56%) of the 180 leukemia patients had sCR1 levels above the normal range (P , 0.001; Fig. 1). The increase in sCR1 is likely to be due to shedding of CR1 from the blast cells, although many alternatives are possible. Liver diseases and direct hepatocyte damage are responsible for an increase in sCR1 [10]. This increase is reverted to normal after liver transplantation. Because hepatocytes do not produce CR1, the most likely explanation is that sCR1 catabolism is reduced in liver failure. Liver dysfunction might have been present in some of the patients analyzed here, although this would not explain the fact that sCR1 is increased in more than half the patients with leukemia. End-stage renal failure is also associated with a small increase in sCR1, the mechanism of this increase being unknown [9]. However, none of the patients described here were on hemodialysis, and the very high levels of sCR1 observed in some patients with leukemia far exceed what is to be expected from a loss of renal function. Finally, it is possible that a tissue or cell type not known to produce CR1 physiologically has been induced to do so under pathological circumstances. The leukemic cells might have induced such a production either by direct contact or via the release of an undefined cytokine. The rather poor correlation between the leukocyte cell counts and sCR1 might be related to several factors. It is well known http://www.jleukbio.org
Fig. 9. Total alternative pathway lytic activity. (A) In normal serum, rsCR1 produced an inhibition of lysis that was reverted by the monoclonal anti-CR1 Ab (3D9); (B) In a T-ALL serum with a very high level of sCR1 (800 ng/mL), the lysis was improved by the addition of monoclonal anti-CR1 Ab (3D9) in a 100-fold molar excess.
that CR1 is a differentiation antigen for myeloid and lymphoid cells [36, 37]. For instance, in the myelocytic lineage, CR1 is expressed in ,1% of neutrophilic myelocytes, 4% of metamyelocytes, 75% of band, and 98% of segmented neutrophils. In the B cell lineage, CR1 is expressed in 15% of large pre-B cells, 40% of small pre-B, 70% of immature B, 99% of mature B cells, and ,2% of plasma cells [36, 37]. Only a small fraction of T lymphocytes expresses CD35, and antigenic stimulation produces an increase in its expression [27, 28]. From the above it is easy to understand that each tumor cell line may express CR1 to a different degree according to maturation and cell lineage. Thus, the absence of a direct correlation between a specific type of leukemia and sCR1 only confirms the heterogeneity of the tumors. There was, however, one exception. The few patients with very high levels of sCR1 in plasma were the T-ALL patients; this was to some extent a surprise. Normally only a minority of T lymphocytes express CR1 and the level of expression is low. Unfortunately, no T cell of the patients with high levels of sCR1 were available to analyze their expression of CR1. We can only postulate potential mechanisms: T cells might release one (or more) factors responsible for the release of sCR1 by other cells. T cells are known to produce a variety of cytokines, which may act as cell activators or growth factors. For example, in vitro IL-2, IL-4, and TNF-a induce an up-regulation of CR1 on granulocytes and monocytes [31]. However, the increased expression of CR1 represents most probably an activation process during which intracellular pools are mobilized and expressed on the cell surface. In vitro, cell activation induced by these cytokines and by other mediators such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), is accompanied by an increase in sCR1 release [7]. Soluble mediators released by T cells (or blast cells) may not only activate cells but also enhance the synthesis of CR1. In vitro, GM-CSF has been shown to delay apoptosis and neutrophils exposed to GM-CSF have more CR1 mRNA than control neutrophils [7, 34]. In vivo, GM-CSF treatment of patients with bone marrow aplasia enhances the expression of CR1 on the circulating neutrophils [34]. Thus it was of particular interest to analyze the effect of G-CSF on sCR1 in normal individuals who served as peripheral stem cell donors. In all, there was a rapid increase in sCR1 levels in plasma. G-CSF enhanced the release of sCR1 probably by several mechanisms: (1) enhanced CR1 synthesis
in each cell, as shown by the increase in CR1 mRNA expression; (2) activation of the cells; and (3) increase of the total number of cells shedding sCR1. Of interest was that G-CSF had an almost identical effect on sL-selectin as on sCR1 levels, suggesting that the synthesis and release of these two molecules are regulated by similar mechanisms in leukocytes. Both molecules are shed by proteolytic cleavage [7, 38]. There is a three-amino-acid sequence homology between the known L-selectin cleavage site [39] and the putative CR1 cleavage site. In addition, the enzyme(s) responsible for the release of sL-selectin have a relaxed sequence specificity [39], which suggests that they may cleave other proteins. However, there are also clear differences in the synthesis and release reactions as attested by the weak correlation between sCR1 and sL-selectin in patients with leukemia. We have recently shown that leukocytes release two different forms of CR1 [18]. The first form corresponds to the shedding of the extracellular domain of the transmembrane molecule (sCR1), whereas the second is still bound to membranes. The most likely explanation for this second form is that leukocytes release vesicles from their membrane, which bear whole transmembrane CR1. Thus, it might be suggested that the increase in sCR1 in leukemia was due in part to the presence of vesicles released by blast cells or stimulated leukocytes. Our different analyses did, however, not identify CR1 bound to vesicles: no significant concentration of transmembrane CR1 was identified in plasma of leukemia patients or of the G-CSF-treated individuals, and almost no CR1 was lost after ultracentrifugation. CR1 is the most potent cofactor for the inactivation of C4b/C3b by factor I. However, in normal plasma the concentration of sCR1 is approximately four orders of magnitude lower than factor H, which acts as the major cofactor for the inactivation of C3b in the fluid phase (sCR1 5 0.15 nM, factor H 5 3 µM). Thus it has been estimated that under physiological circumstances sCR1 plays no role in controlling complement activation. Weisman et al. estimated that srCR1 is at least 100-fold more potent than factor H on a molar basis [6]. The data shown here indicate that sCR1 may be increased more than 10-fold in some patients. For these reasons, we analyzed the effect of small increases in CR1 on complement function in normal serum. Complement was down-regulated in serum by Sadallah et al.
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increasing the sCR1 concentration between 6- and 20-fold, which corresponded to levels observed in leukemia serum. Three different approaches were used to demonstrate the down-regulation, respectively, of the classical pathway, the alternative pathway, and both pathway activations. The mAb 3D9, which is known to block the C3b binding sites of CR1, was a useful tool to demonstrate the reversibility of the inhibition produced by sCR1. It allowed us to demonstrate that sCR1 inhibited alternative pathway function in the plasma of a leukemia patient. Thus, sCR1 is functional in leukemia and it interferes with complement, although to a minor degree. It should be emphasized that the assays performed in vitro are always diluted systems, and thus do not correspond to physiological conditions. Although we do not think that the sCR1 in leukemia interferes significantly with complement function in plasma, it may be that at the site of tumor infiltration the release of sCR1 produces a local down-regulation of complement. These observations suggest as well that pharmacological intervention, such as G-CSF, may be used to up-regulate sCR1, thus complement inhibition in vivo.
ACKNOWLEDGMENTS This work was supported by Swiss National Science Foundation Grant 32-49446.96 and the Roche Research Foundation. The authors thank the participants of the Swiss Group for Clinical Cancer Research (SAKK) and the Hematology Laboratory, Kantonsspital Basel for providing plasma samples and performing immunophenotypic studies. The authors thank Dr. M. Trendelenburg for his assistance with the statistical analysis.
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