Section of Medical Oncology, Department of Medicine and the Rush Cancer Institute, Rush-Presbyterian St Luke's Medical Center, Chicago, IL, USA

Correspondence to: J MD Plate, Section of Medical Oncology, Rush-Presbyterian St Luke's Medical Center, 1653 West Congress Parkway, Chicago IL 60612, USA: Fax: 312 942 3452

^aCurrent address: University of Cincinnati Medical School, Cincinnati, OH, USA

^bCurrent address: University of Illinois at Chicago (UIC), Chicago, IL, USA

Immunologically committed lymphocytes, especially mature, leukemic B cells, proliferate then accumulate without further cell division in chronic lymphocytic leukemia patients (CLL). These mature, leukemic B cells often produce autoantibodies. Under normal circumstances, immunologically committed lymphocytes that are autoreactive are deleted by a programmed cell death mechanism. In CLL cells, these mechanisms appear to be inhibited; therefore, cells accumulate rather than be destroyed. To understand the mechanism by which cell survival is selected over death in CLL cells, we studied the role of 2 integrins and their ligands in the regulation of apoptosis. CLL cells were treated with monoclonal antibodies directed against 2 integrins. Antibodies directed against the I-domain of the chain of CD11b/CD18 inhibited apoptosis. The identity of the physiological ligand or counter-receptor for 2 integrins that was required for the inhibition of apoptosis induction was sought. The ligand iC3b, but not ICAM-1 or fibrinogen, was identified as a ligand that could prevent apoptosis of CLL B cells. Free iC3b levels were elevated in CLL patients indicating that this ligand is available in vivowhere it may interact with 2 integrins on CLL B cells and sustain their viability by preventing activation of the programmed cell death pathway. Leukemia (2000) 14, 34-39.

CLL; apoptosis; 2 integrin CD11b/CD18; iC3b; ICAM-1

Introduction

Chronic lymphocytic leukemia (CLL) is a disease resulting from monoclonal transformation of B cells that belong to a minor CD5⁺ subset often found to be responsible for the production of autoantibodies, B-1.^1,2 The leukemic cells are normally transfixed in a stage of B cell differentiation subsequent to immunoglobulin gene rearrangement but before constant region switching, hence are considered to be relatively mature cells. Although CLL B cells accumulate in patients, most CLL cells are not dividing but appear to be activated with respect to proteins that are tyrosine phosphorylated (Plate et al, unpublished data). CLL B cells express mRNA for a number of growth cytokines, yet are arrested in their growth/death cycle. When isolated and cultured, they do not grow in an autocrine manner, but undergo programmed cell death (PCD), and exhibit classic features of apoptosis.^{3,4,5,6,7,8,9,10,11} The kinetics of the loss of mRNA for one of the cytokines expressed by CLL cells, interleukin 7 (IL-7), coincides with apoptosis induction indicating that degradation of labile mRNA may result during apoptosis induction.¹¹ It has been suggested, in fact, that IL-7 may serve as a viability factor in these cells.^11,12

Genes required for apoptosis appear to be already expressed in CLL cells, but other gene products, such as IL-7, suppress progression of these cells through PCD in vivo.¹¹ Treatment of CLL B cells with actinomycin D, an antibiotic that blocks transcription, or fludarabine phosphate (Fludara) for example, enhances the expression of apoptosis and decreases the time of detection from 18-24 h to 4-6 h. These data suggest that gene transcription is required to maintain CLL B cell viability. We have demonstrated that one mechanism by which gene expression can be maintained and apoptosis of CLL B cells held in abeyance in vitro is through integrin signaling leading us to suggest that ligands presented in the immediate environment of CLL patients may be responsible for maintaining CLL B cell viability.¹³ In this paper we further define the nature of adhesion molecules and ligands that maintain survival of CLL cells. The objective was to define signals for CLL B cells that regulate expression of survival and death proteins and sustain their balance in favor of life.

Materials and methods

Cells

Blood from leukemic patients was obtained through the Hematology Service with Institutional Review Board approval. Mononuclear cells were isolated on a Ficoll-Na Diatrizoate solution as previously reported (Organon Teknika, Durham, NC, USA).¹¹ Cells were frozen in aliquots of 1-3 ´ 10⁷ each. Cells recovered upon thawing were generally from 75-95% viable. Leukemic patients' cells were phenotyped for surface markers by flow cytometry.

Mouse B16 melanoma cell lines, B78H1 and B16 melanoma cells transfected and selected for high expression of human ICAM-1 (UILL/MTX) as well as the human ICAM-1 transfected partner, UIll/G418, were received from Dr Lloyd Graf Jr (UIC, Chicago, IL, USA).¹⁴ These cell lines grow as adherent monolayers. Trysinized cells were seeded into 2 ml well plates at 400 000 cells/well. After overnight culture, CLL cells were added at 1.5 ´ 10⁶ cells/ml in RPMI 1640 medium containing 5% FBC, 2 mM glutamine, and gentamicin. The cultures were further incubated for 24-48 h at 37°C in a 5% humidified atmosphere of CO₂, then CLL cells were collected, solubilized and soluble DNA fragments isolated for analysis of apoptosis.

Reagents

Fludara (fludarabine phosphate) was obtained from Berlex Laboratories, Richmond, CA, USA. Actinomycin D and fibrinogen was obtained from Sigma Chemical, St Louis, MO, USA.

Cell cultures

CLL leukemic B cells were cultured at 1.5-2.0 ´ 10⁶ in 24-well plates in RPMI 1640 supplemented with 2 mM L-glutamine, 5% fetal calf serum, and antibiotics at 37°C in a 5% atmosphere of CO₂. Splenic B-lymphocytes from B10.A mice (Jackson Laboratories, Bar Harbor, ME, USA) were isolated and cultured in RPMI 1640 medium as above.

Antibodies

The hybridomas M1/70 and LM2/1 were purchased from ATCC (Rockville, MD, USA). These anti-11b (CR3) antibodies were purified from supernatant over columns of immobilized protein A (MonoPure-Pierce Chemical, Rockford, IL, USA). Monoclonal anti-iC3b and iC3b ELIZA kits were purchased from Quidel (San Diego, CA, USA). CA-7 anti-ICAM-1 monoclonal antibody was received from R Rothlein (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, USA).

Complement fixation

Normal human serum was diluted to 20% in RPMI 1640 and 250 l of a sterile, rehydrated, dense slurry of Sephadex G10 beads (Pharmacia, Piscataway, NJ, USA) added following established procedures.¹⁵ The mixture was incubated at 37°C for 45 min with frequent gentle mixing to keep the G10 beads in suspension. The beads were then allowed to settle and the serum drawn off. The G10 beads were washed twice in RPMI and finally resuspended in CLL culture medium and 100 l added to each 2.0 ml well with 2 ´ 10⁶ CLL cells. The mixtures were incubated for 24 h before soluble, fragmented DNA was isolated and analyzed to determine the effect of the G10 fixed iC3b on apoptosis.

Apoptosis

Characteristic changes in DNA structure occur during apoptotic cell death due to endogenous endonuclease activity, and is evidenced by a ladder pattern of DNA fragments of increasing size upon electrophoresis.³ Cells were lysed with 0.2% Triton X-100 in Tris-EDTA buffer, pH 8.0, then pelleted. The supernatant containing soluble fragmented DNA was precipitated with cold isopropanol plus 0.5 M NaCl at -20°C, pelleted and washed with 70% ethanol. The precipitate was dissolved overnight in Tris-EDTA-buffer at 37°C. Solubilized DNA was subjected to electrophoresis in a 2.0% agarose gel. The gels were stained with ethidium bromide, visualized on a UV-light box and photographed.

Results

The transcriptional inhibitor, actinomycin D, enhanced the kinetics of apoptosis in cultures of isolated CLL cells while it had little apoptotic effect on peripheral blood mononuclear cells from healthy donors (Figure 1). Apoptosis was readily evident within 4 h of actinomycin D treatment while apoptosis was observed in CLL cells after 24 h of culture alone with medium. These findings demonstrate that proteins required for apoptosis are already expressed in vivo. Evidently protective survival proteins are lost upon culture, and death zymogens (pro-proteins) become activated. Fludara also induced rapid apoptosis in CLL cells (Figure 2). EBV-transformed cells from this same donor, however were resistant to Fludara-induced apoptosis. The fact that Fludara did not induce apoptosis of the EBV-transformed cells and actinomycin D did induce apoptosis of normal cells demonstrates the unique features of the leukemic CLL B cells in that they appear primed for programmed cell death but initiation of the pathway is prevented by labile survival signals including members of the bcl2 family of proteins.¹⁶

One mechanism that holds apoptosis in abeyance is integrin signaling.¹³ We have demonstrated that culture on HUVEC hybrid cells down-regulated apoptosis of CLL cells.¹¹ Furthermore, the expression of CD11b/CD18 has been correlated with the ability of leukemic cells to adhere to human umbilical vein endothelial cells (HUVEC).¹⁷ We examined, thus whether ₂-integrins may be capable of transducing anti-apoptotic signals to the leukemic cells. The data demonstrate that antibodies directed against CD11b (Mac-1) could protect CLL cells from apoptosis (Figures 3 and 4). CD11b encodes the -chain of the Mac-1 adhesion molecule expressed on CD5⁺ B cells. A number of monoclonal antibodies have been raised against CD11b that recognize different epitopes. A survey of these monoclonal antibodies revealed that one denoted LM/2 which recognizes the interactive (I-) or ligand binding region, prevented apoptosis while another monoclonal antibody that recognizes epitopes outside of the I-region, M70/1, did not (Figure 3). These data suggest that signaling via the ligand binding domain of CD11b can prevent apoptosis of CLL cells.

Adhesion molecules are promiscuous with respect to their ligand specificity, hence the identity of ligands capable of binding to CD11b/CD18 that could maintain an anti-apoptotic signal in CLL B cells was sought. ICAM-1 serves as a ligand/counter-reception for CD11b/CD18, and is expressed on a variety of cells including endothelial cells, therefore appeared to be a likely candidate for the molecule that might trigger CLL cell viability, ie suppress apoptosis. Several approaches were undertaken to determine whether ICAM-1 served as the signal presented to CLL cells to prevent apoptosis. Used were (1) monoclonal antibodies (mAb) directed against ICAM-1 to block its possible effect; (2) a lawn of ICAM-1 ligand/counter-receptor was generated by isolation and immobilization of ICAM-1 from human serum on to plastic by capture with the CA-7 mAb (which binds to a domain of ICAM-1 distinct from domains that interact with ₂-integrins), thus fixed it on to plastic such that the active ICAM-1 domains were available to bind to their receptors,¹⁸ and (3) B16 cells transfected with the gene for human ICAM-1 to determine whether surface expressed ICAM-1 could prevent apoptosis of CLL cells. Experiments designed to either block the interactions between CLL cells and EA.hy926 with anti-ICAM mAbs or to stimulate cultured CLL cells with purified soluble ICAM-1 immobilized on to plastic revealed that ICAM-1 did not serve to protect CLL cells from apoptosis (data not presented). Likewise, cells transfected with the human ICAM-1 gene did not protect CLL cells from apoptosis. Apoptosis was observed in CLL cells cultured on both the parental B16 line and the ICAM-1 transfected cells (data not presented). It was concluded from these series of experiments that ICAM-1 did not serve as an anti-apoptotic signal for CLL B cells.

Another counter-reception/ligand, iC3b, is an enzymatically cleaved product that results from the fixation of complement, specifically C3, by antigen-antibody complexes. iC3b serves as a ligand for both CD11b/CD18 and CD11c/CD18.^15,19 In fact, studies have suggested that iC3b can serve as a growth factor for leukemic and normal human B cells.^20,21 Experiments were designed to determine whether iC3b activated ₂-integrins on CLL cells could suppress apoptosis. iC3b from normal donor sera was fixed on to Sephadex G10 beads and incubated with CLL cells.¹⁵ Complement cascade components of C3, specifically iC3b fixed to Sepharose beads, provoked the viability signal and held apoptosis in abeyance (Figure 5, lane 5 vs 4) while culture of CLL cells with soluble or adhered fibrinogen (Figure 5, lanes 1, 2 and 3, respectively), failed to protect CLL cells from apoptosis. Fibrinogen also serves as a counter-reception/ligand for CD11b/CD18.²² These data suggest that activation products of C3 can serve as ligands to maintain CLL viability through 2 integrin transduced signals.

In order to determine whether iC3b might be available in CLL patients to serve as an anti-apoptotic signal and be a factor in CLL cell survival, sera from three control donors and eight CLL patients were assayed quantitatively for iC3b levels by EIA (Quidel). iC3b levels were elevated from 5 to 25 times greater than that found in normal sera (Table 1). Most patients exhibited greater than 10-fold higher iC3b concentrations. These findings indicate that iC3b is readily available for binding to its receptors in CLL patients and may serve as an anti-apoptotic signal in vivo.

Discussion

Adhesion molecules function at different stages of B cell development and protect the maturing B cell against apoptosis. CLL cells are caught in a differentiation stage where they should have been negatively selected against, but failed to have received a disconnect signal mediated through release from adhesion molecules, to undergo apoptosis. Apoptosis of CLL B cells in culture is similar to the behavior of germinal center B cells, known as centrocytes. Centrocytes are relatively mature B cells that have been selected by antigen but whose immunoglobulin genes are undergoing rapid somatic hypermutation, in a process designed to select for high affinity immunoglobulin and memory B cells.²³ These centrocytes normally develop amongst a network of follicular dendritic cells (FDC) that present antigen in the form of immune complexes held on their surfaces by iC3b and Fc receptors for long periods of time. Contact between ₂-integrin and/or VLA-4 expressed on centrocytes and their respective counterreceptors on FDCs is required in order for these B cells to survive.^23,24,25,26 When centrocytes are positively selected for high affinity surface immunoglobulin, they are released from the FDC and survive independently. If released before positive selection, however they undergo apoptosis. Monoclonal antibodies directed against either the adhesion molecules or their ligands/counter receptors inhibit binding (namely between CD11a/CD18 and ICAM-1 or VLA/4 and VCAM), releasing the centrocytes thereby inducing apoptosis.^25,26 CD11b/CD18 (CR3) is also expressed on FDC and is responsible for antigen presentation to the centrocytes via binding of complement fixation components, including iC3b, affixed to immune complexes.²⁷ CLL cells express CD11b^28,29 and the expression of CD11b/CD18 has been shown to be associated with the ability of CLL B cells to adhere to HUVEC cells.^17,28 Our studies have demonstrated that adhesion of CLL B cells to hybrid HUVEC cells prevent apoptosis.¹¹ CD21 or complement receptors type 2 (CR2) are also expressed on FDC and bind iC3b equally as avidly as CR3.³⁰ CD21 is commonly expressed on CLL cells and is present in soluble forms in CLL patients.³¹ Whether or not CD21 can transduce an anti-apoptotic signal in CLL cells is unknown. CD11c/CD18 (CR4 or p150,95) also binds iC3b, and is expressed on cells from most CLL patients (56/70).^32,33 The studies presented here demonstrate that adhesion molecules on CLL cells may play a role in maintaining CLL cell survival in vivo. It was demonstrated that CLL B cells from most patients were responsive to mAbs directed against the A/I domain of CD11b in that they prevented apoptosis, however mAbs directed against CD18 or CD11a were ineffective.

CD11b/CD18 is composed of two chains, the _m-chain (CD11b) and the ₂-chain (CD18). It is one of four members of the ₂-integrin family of adhesion molecules which share in common the ₂-chain (CD18) but each has a distinct -chain gene product.³⁴ Each alpha chain is composed of seven homologous tandem repeats of 60 amino acids in length and a relatively large (187 amino acids) I-domain (interactive) separating the second and third repeats. The I domain also has a significant homology to a similar domain found in the complement proteins C2 and factor B, the latter of which binds to C3b. The A/I domain is the major recognition site on CD11b for iC3b, fibrinogen, and ICAM-1.³⁵ The A/I domain seems to be the major interactive portion of the molecule but ligand binding generally requires 'activation' of the heterodimer. Activation of integrins results in a change in their affinity of binding from a low affinity state to a high affinity state.³⁶ Monoclonal antibodies have been developed that recognize different domains and activation states of the integrins. In addition, some mAbs can 'activate' binding affinity changes of the integrins by mechanisms that are still not fully understood. The dynamic interactions of adhesion molecules on cell surfaces greatly affect the affinity of receptor-ligand interactions and can determine whether binding and subsequent signaling via second adhesion molecules in a cascade will occur. Some mAbs directed against the binding site can also transduce signals. Our data demonstrate that monoclonal antibodies directed against the _m-chain of the ₂-integrin, CD11b/CD18, transduce a viability signal to CLL and normal mouse B cells (Figures 3 and 4). Two types of mAbs directed against CD11b were tested to determine whether they could directly activate an anti-apoptotic signal: one type recognizes the A/I domain while the other does not.²² The M1/70 mAb which does not recognize an epitope found in the A/I domain had no direct affect on CLL B cell apoptosis. The LM2/1 mAb, however, which does recognize an A/I domain epitope does prevent apoptosis of CLL B cells. These data suggest that the A/I domain of CD11b may be capable of transducing an anti-apoptotic signal to CLL B cell to promote survival thereby prevent apoptosis.

A number of natural ligands for CD11b/CD18 have been described and were tested.^{15,35,37,38,39,40} Neither ICAM-1 nor fibrinogen inhibited apoptosis of CLL B cell apoptosis, however incubation with iC3b did result in inhibition. iC3b, therefore, may serve as the natural anti-apoptotic ligand in CLL patients. iC3b is a relatively stable and natural enzymatic activation product of the C3 component of complement. Infectious organisms and antibody fixation can result in C3 activation. Since CLL leukemic B cells are usually CD5⁺, they may secrete sufficient quantities of autoreactive antibodies which upon binding to antigen would fix complement thereby increasing the levels of iC3b.^20,33,41 Disease progression in CLL patients could then be augmented by iC3b if iC3b served as the active ligand for C11b/CD18 to signal growth and/or viability of CLL cells. In fact, iC3b levels in CLL patients were on average >14 times higher than normal (Table 1). iC3b is also recognized as a regulator of antibody responses; first, by localizing with immune complexes on to follicular dendritic cells of germinal centers hence, serving to promote centrocyte proliferation and selection for B cells with high affinity immunoglobulin receptors, and second, by directly serving as a polyclonal activator of B cells.²⁰ Plasma levels of iC3b are generally elevated during infections, in autoimmune diseases, and as demonstrated here, in CLL patients. Furthermore, Hy.EA926 and human umbilical vein endothelial cells (HUVEC) express Factors H and I which convert the activated complement component C3 into iC3b, hence their support of CLL B cell survival may be attributable to complement activation by-products.⁴² A reduction of the leukemic load in patients then could serve two benefits, clearance of tumor, as well as reduction of iC3b levels. As a polyclonal activator of B cell growth, decreased quantities of iC3b in these patients would also limit proliferation signals for leukemic cells growth.

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Figure 1  Actinomycin D treatment of CLL cells leads to more rapid expression of apoptosis while similar treatment of normal donor cells resulted in no visible apoptosis. CLL cultures with actinomycin D exhibited apoptosis as early as 6 h while apoptosis of CLL cells cultured with medium was first clearly apparent after 24 h in culture. Normal donor cells cultured with 50 ng/ml actinomycin D for: lane 1, 2 h; lane 2, 4 h; lane 3, 24 h. CLL leukemic cells were either cultured alone, with 50 ng/ml actinomycin D or 10 ng/ml actinomycin D, respectively in lanes 4, 5, 6: for 2 h; respectively in lanes 7, 8, 9: for 4 h; respectively in lanes 10, 11, 12: for 6 h; and respectively in lanes 13, 14, 15: for 24 h. DNA was isolated, separated by gel electrophoresis in 0.8% agarose gel, stained with ethidium bromide, visualized on a UV-light source and photographed.

Figure 2  Leukemic cells from CLL patients are more sensitive to apoptotic induction in vitro than are other transformed cell types. CLL cells treated with Fludara in vitro were readily induced to undergo apoptosis (lane 3), without Fludara (lane 4). Similarly treated EBV-transformed cells established from a CLL patient, however do not show evidence of apoptosis with Fludara (lane 2), without Fludara (lane 1). DNA was isolated, separated in 1.0% agarose gel electrophoresis, stained with ethidium bromide, visualized on a UV-light source and photographed.

Figure 3  Monoclonal antibody, LM2/1, directed against the CD11b A/I domain inhibited apoptosis of CLL B cells. CLL cells were cultured with two mAbs directed against the alpha chain of CD11b, one directed against the A/I domain, LM2/1; the other directed outside the A/I domain, M1/70. DNA was isolated, separated in 0.8% agarose gel, stained with ethidium bromide, visualized on UV-light and photographed. CLL cells were incubated with mAb HB204 (LM2/1 anti-MAC-l) in lane 1; TIB128 (M1/70 anti-MAC-l) lane 2; and medium alone, lane 3.

Figure 4  Monoclonal antibody directed against the -chain, CD11b, prevents apoptosis of normal B10. A mouse splenic B cells after 24 h in culture, while mAbs against CD11a, CD18 and VLA-4 do not. Lane 1, cells cultured without mAb; lane 2, anti-VLA-4 mAb; lane 3, anti-CD11a -chain mAb (LFA-1); lane 4, anti-CD11b -chain mAb (CR3), and lane 5, anti-CD18 -chain mAb. Apoptosis was readily observed in all mouse splenic B cell cultures except those incubated with mAb directed against the -chain of CD11b/CD18 ₂-integrin (CR3/Mac-1). (These data were collected as part of a collaborative study with Drs K Siemasko and A Finnegan, Section of Rheumatology, Rush-Presbyterian St Luke's Medical Center).

Figure 5  Complement components from normal human serum activated on Sephadex beads serve as anti-apoptotic signals for CLL B cells while another CD11b/CD18 ligand, fibrinogen does not. CLL cells were culture with: lane 1, 250 g soluble fibrinogen; lane 2, 500 g soluble fibrinogen; lane 3, 500 g fibrinogen prefixed to plastic bottom of well; lane 4, Sephadex G10 beads alone; lane 5, Sephadex G10 beads preincubated at 37°C, 45 min, in normal donor human serum; lane 6, Sephadex G10 preincubated in serum from CLL donor; lane 7, fresh CLL cells exhibiting no apoptosis; and lane 8, CLL cells cultured alone without ligands exhibit apoptosis.

Table 1  Elevated iC3b levels in plasma or sera obtained from CLL patients