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The Journal of Immunology, 2000, 164: 6075-6081.
Copyright © 2000 by The American Association of Immunologists

Exceptional Resistance of Human H2 Glioblastoma Cells to Complement-Mediated Killing by Expression and Utilization of Factor H and Factor H-Like Protein 11

S. Junnikkala*, T. S. Jokiranta*, M. A. Friese{dagger}, H. Jarva*, P. F. Zipfel{dagger} and S. Meri2,*

* Department of Bacteriology and Immunology, Haatman Institute, University of Helsinki, Helsinki, Finland; and {dagger} Research Group for Biomolecular Medicine, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The resistance of human cells, including those in malignant tumors, to complement-mediated damage is mainly mediated by specific membrane inhibitors of complement (1, 2). These regulators include complement receptor 1 (CR1;3 CD35), membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55), and protectin (CD59). CR1 and MCP act as cofactors for factor I (C3b inactivator)-mediated degradation of C3b (3, 4), while CR1 and DAF inhibit the assembly and promote the decay of the C3/C5 convertases of both the classical and the alternative complement pathways (3, 5). CD59 inhibits formation of the membrane attack complex on the surface of homologous cells (6, 7, 8).

In addition to the membrane regulators, complement activation is efficiently controlled in the fluid phase by factor H, which is synthesized mainly by the liver (9), monocytes, and macrophages (10, 11). Factor H normally participates in the inactivation of soluble C3b (12). Plasma factor H has all of the regulatory activity at the alternative pathway C3 convertase level by being a cofactor for factor I-mediated cleavage of C3b to iC3b, competing with factor B for binding to C3b and by dissociating Bb away from the C3bBb convertase complex (13, 14, 15). In addition to restricting alternative complement pathway activation in the fluid phase, factor H down-modulates complement activation on host cells by simultaneous recognition of C3b and sialic acids or glycosaminoglycans on cell surfaces (16, 17, 18). Binding of factor H to polyanions has been shown to be mediated by short consensus repeat (SCR) domains 7 and 20 (19) and possibly by a region near SCR 13 (20). On structures that lack the membrane regulators DAF and MCP, like the basement membranes in kidney glomeruli, the protection depends on factor H (21). As an illustrative example, deficiency of factor H has been shown to lead to membranoproliferative complement-mediated glomerulonephritis both in humans (22) and in pigs (23).

During our studies on the expression of complement regulators in human gliomas, we found that the human H2 glioblastoma cell line was exceptionally resistant to complement-mediated lysis (24). Most nucleated cell lines growing in vitro can be sensitized to complement killing by neutralization of the membrane regulators with appropriate mAb (25, 26, 27). However, in the case of H2 cells, not even a combined neutralization of MCP, DAF, and CD59 appreciably increased the sensitivity of the cells to complement killing. This suggested that the H2 cells may utilize an additional, hitherto undefined, mechanism to resist complement-mediated cytotoxicity. The purpose of the present study was to examine this mechanism.

We observed that the H2 cells themselves synthesized and secreted factor H, as well as the factor H-like protein 1 (FHL-1), a truncated but functionally active form of factor H composed of seven (of 20) N-terminal SCRs of factor H (28, 29). FHL-1 is a product of alternative splicing of the factor H gene and has essentially the same functions (cofactor activity and decay-accelerating activity) as factor H (28, 30, 31). FHL-1 has also been suggested to have an additional role in cell adhesion and is found in human plasma at a concentration 10–50 times lower than that of factor H (29). Because FHL-1 has the heparin binding site within SCR 7, it has the potential to bind to cell surface glycosaminoglycans and/or sialic acids.

We also found out that factor H synthesized or recruited from the fluid phase could bind to the H2 cell membrane. Neutralization of factor H and FHL-1 with appropriate mAb was the most efficient way to sensitize H2 cells to complement-mediated killing. Also, removal of sialic acids resulted in increased lysis of the H2 cells by the alternative pathway of complement. The results show that endogenous production and/or binding of soluble complement inhibitors factor H and FHL-1 to the cell membrane are mechanisms that malignant cells can utilize to protect themselves against complement-mediated killing.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

The H2 glioblastoma cell line displaying features of the giant cell variant was derived from a female patient who died because of a rapidly expanding local brain tumor (32). The human endothelial-like (EA.hy 926) and U251 glioma cell lines were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were grown in RPMI 1640 medium (Life Technologies, Paisley, U.K.) supplemented with 10% (v/v) heat-inactivated FCS (Life Technologies), penicillin (10 U/ml), streptomycin (100 µg/ml), and 2 mM L-glutamine (Nord Cell, Bromma, Sweden).

Abs and other reagents

Rabbit polyclonal anti-H2 antiserum was raised by immunizing a rabbit twice (i.m.) with 107 heat-killed H2 glioma cells. Anti-CD59 mAb YTH53.1 (rat IgG2b) was purified from the hybridoma cell growth medium supernatant using a Sepharose protein G column (Pharmacia, Uppsala, Sweden), as described (33). The cell clone was originally obtained from Dr. H. Waldmann (Sir William Dunn School of Pathology, University of Oxford, U.K.). The anti-CD59 mAb BRIC229 (mouse IgG2b) and anti-CD55 mAb BRIC216 (mouse IgG1) were purchased from Bio-Products Laboratory (Elstree, U.K.). The mouse anti-CD46 mAb GB24 (IgG1) was kindly obtained from Dr. K. Liszewski and Dr. J. P. Atkinson (Washington University, St. Louis, MO), and anti-CD46 mAb 10L46 was from Immunotech S.A. (Marseille, France). Cell lines producing the anti-factor H mAbs 131X, 90X, and 196X were kindly obtained from Dr. J. Tamerius (Quidel, San Diego, CA) (34), and OX24 mAb was from Dr. R. B. Sim (MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford). Polyclonal anti-factor H Ab was from Incstar (Stillwater, MO). Anti-CD35 Ab was obtained from Becton Dickinson (San Francisco, CA). Anti-iC3b and anti-C5b-9 (Wu 7.2) mAb were from Quidel and Dr. R. Würzner (Innsbruck, Austria), respectively. Heparitinase (type III), neuraminidase (type V from Arthrobacter ureafaciens), trypsin, and phosphoinositol-specific phospholipase C (PIPLC) (from Bacillus cereus) were obtained from Sigma (St. Louis, MO). The cysteine proteinase inhibitor E-64 was from Boehringer Mannheim (Mannheim, Germany).

Flow cytometry

For flow-cytometric analysis, H2, U251, and EA.hy 926 cells were detached from culture dishes with 0.02% EDTA, washed twice, and suspended into 0.5% BSA/PBS. For each staining, 5 x 105 cells were incubated (30 min on ice) with the primary mAb. When iC3b (10 µg/ml) and C5b-9 (10 µg/ml) depositions were studied, the cells were first treated with the complement-activating YTH53.1 mAb (50 µg/ml) and NHS (diluted 1/4) for 30 min at 37°C, washed twice, and then incubated for 30 min on ice with the primary mAb (10 µg/ml). After washing the cells twice, they were incubated for an additional 30 min on ice with FITC-conjugated rat anti-mouse or goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), washed again twice, and then analyzed on a Becton Dickinson FACScan 440 flow cytometer (San Jose, CA). To analyze the effect of PIPLC treatment on GPI-anchored proteins CD55 and CD59, H2 cells were incubated with or without PIPLC (0.5 IU/ml) for 60 min at 37°C, washed, and incubated with anti-CD55 (BRIC216) or anti-CD59 (BRIC229) mAb (both at 10 µg/ml) for 30 min at 4°C. After washing, the cells were incubated for an additional 30 min at 4°C with FITC-conjugated rabbit anti-mouse IgG, washed, and analyzed on the flow cytometer.

In experiments in which binding of factor H to cell membranes was studied, the cells were first incubated for 1 h at 37°C with NHS (diluted 1/4) or with purified factor H (200 µg/ml) for 30 min at 37°C. Following a 30-min incubation with polyclonal anti-factor H Ab (diluted 1/50) at 4°C, the cells were washed carefully and incubated with the secondary Ab (FITC-conjugated rabbit anti-goat IgG) for 30 min at 4°C, washed, and analyzed. Control stainings were performed using an irrelevant mouse or rabbit IgG as the primary Ab.

Binding of radiolabeled factor H to H2 and U251 cells

Complement factor H was purified as described (17, 18) and radiolabeled with Na125I (NEN, Boston, MA), according to the manufacturer’s instructions. Free 125I was separated from the labeled proteins by gel filtration through Sephadex G25 (Pharmacia). Initial sp. act. of the radiolabeled factor H was 3 x 106 cpm/µg.

Binding of factor H to cells was examined by incubating triplicate samples of 3 x 105 of H2 or U251 cells with radiolabeled factor H (1-100 ng) for 60 min at 37°C with continuous shaking in a final volume of 120 µl of GVBS (0.1% gelatin, veronal-buffered saline, pH 7.4) diluted 1/3. In inhibition experiments, varying concentrations (0.003–30 µg/ml) of unlabeled factor H or BSA were mixed with 30 ng of 125I-labeled factor H and incubated with cells for 60 min at 37°C with continuous shaking. The mixture (100 µl) was layered on top of a 250-µl column of 20% sucrose in narrow 0.4-ml test tubes and centrifuged for 5 min at 6100 x g. Cell pellets were cut off from the tubes. Both pellets and supernatants were counted for radioactivity, and the binding percentages were determined as the proportion of cell-bound vs total radioactivity. Experiments were performed in duplicate. Binding properties of factor H to H2 cells were examined by subjecting the data to Scatchard analysis.

Complement-mediated lysis of cells

The sensitivity of cells to complement lysis was determined, as described earlier (24). Briefly, cells (2 x 106) were labeled with 100 µCi of 51Cr (Amersham, Amersham, U.K.) in 1 ml RPMI containing 10% FCS for 2 h at 37°C, washed twice with RPMI, incubated for an additional 30 min to remove loosely bound 51Cr, and washed twice. Duplicate aliquots of 51Cr-labeled cells (105 cells/50 µl) were treated with appropriate Abs (20 min, 22°C) and NHS (30 min, 37°C) in a total volume of 200 µl. When the effects of PIPLC, neuraminidase, and trypsin were examined, the cells were first incubated for 1 h at 37°C with the reagents at concentrations described in the respective figure legends. The effect of factor H neutralization on complement-mediated killing of H2 cells was studied by treating the cells with the complement-activating and CD59-neutralizing Ab (YTH53.1) alone or in the presence of the anti-factor H mAb 131X (50 µg/ml) or a mixture of 131X and another anti-factor mAb, OX24 (both at 50 µg/ml). The amount of cell lysis was calculated on the basis of 51Cr released, as described (24). All experiments were performed in duplicate and repeated at least once.

RT-PCR analysis

Total RNA was extracted from the cells using Trizol LS Reagent (Life Technologies), as recommended by the manufacturer. RNA from human liver tissue was isolated with guanidine thiocyanate and purified by CsCl centrifugation (35). Isopropyl alcohol-precipitated RNA was dissolved in diethylpyrocarbonate-treated water, and 1 µg of RNA was denaturated at 70°C for 10 min and then immediately chilled on ice. Reverse-transcriptase reactions were conducted using RNA in a total volume of 20 µl containing 10 mM DTT (Life Technologies), 500 µM dNTP mix (Pharmacia), 25 µg/ml oligo(dT)12–18 (Pharmacia), and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies) in a reverse-transcriptase buffer (Life Technologies). The reaction was allowed to occur for 1 h at 37°C.

Each PCR was conducted in a 100 µl volume containing 200 µM dNTP (Pharmacia), 10 pmol of each of the specific primers, and 2.5 U Taq polymerase (Pharmacia) in an appropriate PCR buffer containing 1.5 µM MgCl2. The samples were denatured at 94°C for 5 min, and amplification was performed on a Perkin-Elmer (Norwalk, CT) GeneAmp PCR System 2400, with denaturation at 94°C for 1 min, annealing at 46°C for 1 min, and extension at 72°C for 1 min for 30 cycles. The final reaction step was followed by a 10-min extension step at 72°C to ensure that the amplified DNA was double stranded. To confirm the absence of contaminants, negative controls were included in each RT-PCR assay, in which the RNA samples were replaced by sterile water or the reverse transcriptase was omitted. Amplified products (10 µl for each PCR sample) were electrophoresed in parallel with size markers (Life Technologies) on a 1% agarose gel. The gels were stained with ethidium bromide and photographed under UV light. The identities of the PCR products were confirmed by cloning into pCRII TA cloning vector (Invitrogen, Groningen, The Netherlands) and sequence analysis.

Reverse-transcribed mRNA was amplified by PCR using the following (sense/antisense) primers at the indicated position of each PCR product: 5'-CTTCCTTGTAAATCTCCACCTG-3', 5'-TCTGCATGTTGGCCTTCCTGTC-3', 2861–3215 (factor H); 5'-TACTGGCTGGATACCTGCTCCG-3', 5'-CAGAAGTTCAGAGGGTAAAGCT-3', 1006–1430 (FHL-1); 5'-TGTGAGGAGCCACCAACATTTG-3', 5'-TTGGGGGCTTACGGCTCCAAAT-3', 137–500 (CD46); 5'-TGACTGTGGCCTTCCCCCAGAT-3', 5'-GTGTTACATGAGAAGGAGATGG-3', 168–640 (CD55); 5'-CTGCAGTGCTACAACTGTCCTA-3', 5'-GGGATGAAGGCTCCAGGCTGCT-3', 139–447 (CD59); 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', 5'-CTAGAAGCATTGCGGTGGACGATGGAGGG-3', 478-1128 (ß-actin).

Immunoblotting and ELISA

H2, U251, and EA.hy 926 growth supernatants (from cells grown in RPMI without FCS for 48 h) were concentrated 50-fold with the Millipore Ultrafree-15 Centrifugal Filter Device (Bedford, MA). Aliquots of concentrated supernatants were electrophoresed on a 10% SDS-PAGE slab gel under nonreducing conditions and transferred to a nitrocellulose filter with a pore size of 0.25 µm (Schleicher & Schuell, Dassel, Germany), according to the method of Towbin et al. (36). After blocking nonspecific binding sites with 5% human milk/PBS, the filter was incubated with an anti-factor H mAb (196X; 10 µg/ml) overnight at 4°C, washed, and incubated with peroxidase-conjugated rabbit anti-mouse IgG (diluted 1/1500; Jackson ImmunoResearch) for 1 h at 22°C. Filters were washed twice, and bound Abs were visualized using an ECL Western blotting kit (Amersham).

For factor H/FHL-1 ELISA, wells in microtiter plates were coated with a polyclonal goat anti-human factor H IgG (2.5 µg/ml) overnight at 4°C. After blocking with 3% BSA/PBS, growth supernatants (cells grown for 4 h without FCS) were added and incubated for 2 h at 37°C. After washing, the 90X mAb (1 µg/ml), which binds to SCR 1 of both factor H and FHL-1, was added. The plates were incubated for 2 h at 37°C and washed, and the bound mAb binding to SCR 1 of both factor H and FHL-1 was detected with a 1/2000 dilution of HRP-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch). Following incubation with the substrate for HRP (Life Technologies), the color reaction was stopped with H2SO4 and the absorbance was measured at 492 nm.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The expression of complement-regulatory proteins on H2 cells

The cell surface expression of complement-regulatory proteins on H2 cells was studied by FACS analysis after staining the cells with appropriate mAb followed by FITC-conjugated anti-mouse IgG. H2 cells showed a strong expression of CD59 (protectin) and a moderate expression of CD46 (MCP) and CD55 (DAF), but no CD35 (CR1) (Fig. 1Go).



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  		FIGURE 1. Flow-cytometric analysis of expression of complement-regulatory proteins CR1 (CD35), MCP (CD46), DAF (CD55), and protectin (CD59) on H2 glioblastoma cells. The cells were stained with 10 µg/ml of the mouse mAb against CD35, CD46 (10L46), CD55 (BRIC216), and CD59 (BRIC229), respectively (hatched histograms). Irrelevant mouse IgG was used as a primary Ab for negative control stainings (open histograms).

 
Complement-mediated lysis of H2 and EA.hy 926 cells

We next compared the complement sensitivity of H2 cells with that of endothelial-like EA.hy 926 cells and another glioma cell line (U251). When treated with a polyclonal anti-H2 Ab (diluted 1/10), anti-CD59 mAb (50 µg/ml), and NHS (diluted 1/4), only 5% of H2 cells became killed (Fig. 2GoA). Under similar conditions, both 251 and EA.hy 926 cells (Fig. 2GoA) were efficiently killed. This result indicated that H2 cells have an exceptional capability to resist complement-mediated killing. However, when CD59 was not neutralized, EA.hy 926 and U251 cells also remained quite resistant to killing (10% and 15% lysis, respectively). To further study the resistance of H2 cells, we inactivated the complement regulators CD46, CD55, and CD59 with specific mAb. This treatment increased lysis only minimally (Fig. 2GoB), showing that these complement regulators are not enough to account for the complement resistance of H2 cells.



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  		FIGURE 2. The effect of complement regulator neutralization on complement-mediated killing of H2, U251, and EA.hy 926 cells. A, The three cell lines were treated with the polyclonal anti-H2 Ab in the absence or presence of the anti-CD59 mAb YTH53.1. B, H2 cells were treated with the polyclonal anti-H2 Ab without ({circ}) or with a mixture of anti-CD59 (YTH53.1), anti-CD46 (GB24), and anti-CD55 (BRIC216) mAbs (•). NHS diluted 1/4 served as the complement source. Cell lysis was detected using a standard 51Cr release assay. Results from two experiments are shown.

 
Because of the exceptional complement resistance of H2 cells, we next tested whether cell surface molecules were responsible for this effect. To this end, we treated H2 cells with B. cereus PIPLC (0.5 IU/ml), which removed 60% and 69% of the GPI-anchored proteins CD55 and CD59, respectively, when examined by FACS analysis. This treatment increased the sensitivity of the cells to lysis in the presence of anti-H2 Ab only by 4% (Fig. 3GoA). Furthermore, treatment of the H2 cells with heparitinase or the cysteine proteinase inhibitor E-64 had no effect on the cell lysis (data not shown). In contrast, treatment of H2 cells with A. ureafaciens neuraminidase increased the cell lysis (Fig. 3GoB), and trypsin treatment efficiently sensitized the cells to complement-mediated killing (Fig. 3GoC). This observation suggested that cell surface structures, such as sialic acid, contribute to the complement resistance of the H2 cells.



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  		FIGURE 3. The effect of treatment with PIPLC (A), neuraminidase (B), or trypsin (C) on complement lysis of H2 cells. A, The cells were first treated with B. cereus PIPLC (0.5 IU/ml) (•) or growth medium ({circ}) for 60 min at 37°C and then with the polyclonal anti-H2 Ab for 20 min at 22°C and NHS for 30 min at 37°C (diluted 1/4). B, The cells were treated with the indicated concentrations of A. ureafaciens neuraminidase for 30 or 60 min at 37°C before the killing assay. C, The cells were first treated with trypsin for 60 min at 37°C and then with the polyclonal anti-H2 Ab (diluted 1/10) and NHS (•) or NHS alone as a control ({circ}). In all cases, lysis was determined by the 51Cr assay. The curves show results from two experiments performed in duplicate.

 
Complement activation on the surface of H2 cells is restricted at or beyond the C3 stage

To analyze the level in which complement activation on H2 cells was arrested, we examined C3b degradation on the H2 cells and analyzed iC3b and C5b-9 neoepitope generation by FACS. First, complement was activated on the surface of H2 cells with the YTH53.1 mAb using NHS as the complement source. In H2 cells and the control cell lines (U251 and EA.hy 926), iC3b and C5b-9 deposits were detected. Moreover, approximately two and three times more C5b-9 neoepitopes were detected on EA.hy 926 and U251 cells, compared with H2 cells (Fig. 4Go). On the other hand, H2 cells converted C3b considerably more efficiently to its inactive form iC3b, as indicated by a 5–6-fold higher expression of the iC3b neoepitope on H2 cells than on the other cell lines tested. This result suggests that H2 cells, at least partly, circumvent complement activation at the C3 level.



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  		FIGURE 4. Flow-cytometric analysis of deposition of the complement iC3b and C5b-9 neoepitopes on H2, U251, and EA.hy 926 cells after serum complement activation (30 min, 37°C) with the YTH53.1 mAb (50 µg/ml). The cells were stained with the mouse mAb against iC3b and C5b-9 (hatched histograms). Irrelevant mouse IgG was used as a primary Ab for the negative control stainings (open histograms).

 
RT-PCR analysis of factor H and FHL-1 mRNA expression by H2 cells

Since the previous experiments suggested a possible role for factor H in the complement resistance of H2 cells, we next examined directly the possibility that H2 cells themselves produced factor H. Expression of the mRNAs of the complement regulator proteins factor H, FHL-1, CD46 (MCP), CD55 (DAF), and CD59 was analyzed in the H2 and U251 glioma cells by RT-PCR. The most striking result was that factor H and FHL-1 mRNAs were clearly detectable in the complement-resistant H2 cell line, but not in the complement-sensitive U251 cell line (Fig. 5GoA). We also examined the EA.hy 926, T47D breast carcinoma (37), and Paju neuronal (38, 39) cell lines, but they were all negative for both factor H and FHL-1 mRNA expression (data not shown). Also, the expression of CD55 and CD59 mRNAs was slightly stronger on the H2 cell line, whereas no clear difference in MCP mRNA expression between the two cell lines was observed. The levels of factor H and FHL-1 mRNAs were comparable with those in the human liver tissue, which serves as the major source for plasma factor H and FHL-1 in vivo.



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  		FIGURE 5. A, RT-PCR analysis of mRNA expression of complement regulators factor H, FHL-1, CD46, CD55, and CD59 in liver and in the complement-resistant (H2) and sensitive (U251) glioma cells. mRNA was isolated from H2 cells, U251 cells, and human liver tissue, as described in Materials and Methods, and analyzed by RT-PCR using specific primers. Actin-ß was used as a positive control. B, Immunoblotting analysis of factor H and FHL-1 secretion. The H2 and U251 glioma cells and the endothelial-like EA.hy 926 cells were grown for 48 h in the absence of FCS. The growth supernatants were collected, concentrated 50-fold, and analyzed by SDS-PAGE and immunoblotting under nonreducing conditions using the 196X mAb (10 µg/ml), which detects both factor H and FHL-1. NHS (1%) served as a positive control. Bound Ab was visualized with a peroxidase-conjugated rabbit anti-mouse Ab as a secondary Ab. Molecular weights of the marker proteins are indicated on the left. Note a higher relative ratio of FHL-1 to factor H in the H2 supernatant as compared with that in serum.

 
Factor H and FHL-1 protein synthesis by H2 cells

As we found that H2 cells expressed factor H and FHL-1 mRNAs, we wanted to analyze the expression also at the protein level. We performed ELISA and immunoblotting analyses from growth supernatants of H2, U251, and EA.hy 926 cell lines. To avoid potential growth medium contamination, the cells were washed extensively and grown in RPMI in the absence of FCS. In ELISA using the anti-factor H mAb 131X and anti-factor H/FHL-1 mAb 90X, the H2 cells, but not the U251 cells, were found to produce both factor H and FHL-1 into the growth supernatant even after a short period (4 h) of incubation (data not shown). Using isolated factor H as a standard, the amount of factor H/FHL-1 produced was estimated to be above 2 ng/ml/105 cells.

By immunoblotting with the 196X mAb, which detects both factor H and FHL-1 by binding to the most N-terminal SCR of both molecules (34), H2 cells were shown to produce both the 150-kDa factor H and the 42-kDa FHL-1 protein (Fig. 5GoB). It appeared that higher amounts of FHL-1 than factor H were produced by the H2 cells. In comparison, in serum the level of factor H is known to be much higher than that of FHL-1 (29). We detected factor H and FHL-1 proteins already after a 24-h incubation (data not shown), but clearly more factor H and FHL-1 were produced after a 48-h incubation. Control cells U251 and EA.hy 926 did not produce detectable amounts of factor H or FHL-1 during the same 48-h incubation period. However, if the cells were incubated without FCS for a prolonged time (7 days), a low amount of factor H and FHL-1 was found also from the growth supernatant of U251 cells, but not from that of EA.hy 926 cells (data not shown).

Binding of factor H to the surface of H2 and U251 cells

Because H2 cells secreted factor H into the growth supernatant and efficiently promoted C3b degradation into iC3b (Fig. 4Go), we wanted to see whether H2 cells directly bound factor H to their cell surfaces. H2 and U251 cells were preincubated first with NHS or purified factor H (200 µg/ml), and after washings with a polyclonal anti-factor H Ab. FACS analysis showed that factor H bound more efficiently to H2 than U251 cells (Fig. 6GoA). Without serum or factor H preincubation, no binding of the anti-factor H Ab to H2 or U251 cells was seen.



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  		FIGURE 6. Factor H binding to glioma cells. A, Flow-cytometric analysis of binding of purified factor H and factor H from NHS to H2 and U251 cells. Cells were first incubated with NHS (1 h at 37°C) or with the purified factor H (200 µg/ml) for 30 min at 37°C and after washing with the polyclonal anti-factor H Ab (50 µg/ml) and FITC-labeled rabbit anti-goat Ab (hatched histograms) at 4°C, as described in Materials and Methods. Irrelevant rabbit IgG was used as a primary Ab for negative control stainings (open histograms). B, Binding of 125I-labeled factor H to H2 and U251 glioma cells. Cells (3 x 105) were incubated with indicated amounts of radiolabeled factor H (3 x 106 cpm/µg) for 60 min at 37°C in 1/3 GVBS, pH 7.4, and then cell-bound and free radioactive proteins were separated by centrifugation of the mixtures through 20% sucrose column. The results are expressed as percentage of bound radioactivity. A representative of two experiments is shown. Scatchard analysis of factor H binding to H2 cells is shown on the right side panel. C, Inhibition of 125I-labeled factor H (30 ng) binding to H2 cells with unlabeled factor H or BSA. Mixtures of labeled and unlabeled proteins were incubated with H2 cells for 60 min at 37°C in 1/3 GVBS and analyzed, as in B.

 
To further examine the nature of factor H binding to H2/U251 cells, dose-response analyses (Fig. 6GoB) and inhibition experiments (Fig. 6GoC) were performed. When H2 and U251 cells were incubated with increasing amounts of 125I-labeled factor H (1–100 ng), the H2 cells were found to bind approximately twice the amount of 125I-labeled factor H that bound to the U251 cells in a saturable manner (Fig. 6GoB). Scatchard analysis of the binding data suggested that factor H bound to the H2 cells in a heterogenous manner (Fig. 6GoB). Binding of 125I-labeled factor H (30 ng) to H2 cells could be inhibited with unlabeled factor H (50% inhibition at 1 µg/ml), but not with BSA (Fig. 6GoC). These results suggest that H2 cells not only produce factor H, but also have the ability to bind soluble factor H from the fluid phase.

The effect of anti-H mAbs on complement-mediated lysis of H2 cells

As the protective effects of sialic acid (removed by neuraminidase) and proteins carrying sialic acid (removed by trypsin) (Fig. 3Go) were presumed to be due to recruitment of the complement regulators factor H and FHL-1 from the fluid phase, we next analyzed the effect of factor H neutralization on complement lysis of H2 cells. For neutralization experiments, we used two Abs. The 131X mAb binds to factor H, but not to FHL-1, and inhibits partly binding of factor H to C3b (34). The OX24 mAb binds to SCR 5 of both factor H and FHL-1 and inhibits partly binding of factor H/FHL-1 to C3b. When treated with the YTH53.1 mAb and 131X mAb, the lysis of H2 cells increased 3-fold (from 5% to 15%), and a combination of the two anti-factor H mAbs (131X and OX24) with the YTH53.1 mAb increased the lysis of H2 cells 5-fold (from 5% to 25%) (data not shown). This indicated that factor H and/or probably FHL-1 had a significant role in the complement resistance of the H2 glioblastoma cells.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have determined a novel mechanism whereby the malignant tumor cell line H2 can resist complement killing. H2 cells were obtained from a case of aggressive glioblastoma. These cells, previously observed to be exceptionally resistant to complement (24), were found to synthesize and secrete the complement regulators factor H and FHL-1. In addition, the cells bound factor H to their cell surfaces and promoted cleavage of cell membrane-deposited C3b to its inactive form iC3b more efficiently than other cell types. As a consequence, the deposition of C5b-9 was reduced, and complement-mediated damage of H2 cells was prevented.

To date, little evidence for the role of factor H in the resistance of tumor cells against complement damage has been presented, and the present study is the first one to propose such a role for FHL-1. However, a recent study suggested for factor H a role into regulating classical pathway activation on the surface of nucleated cells (40). An earlier study by Malhotra et al. demonstrated that the human U937 monocytic cells expressed factor H, which remained associated with the cell surface (41). In addition, the studies of Gasque et al. have shown that certain glioma and neuroblastoma cells express factor H mRNA (42, 43). These authors also reported that a human oligodendrocyte cell line (HOG) secreted factor H, but the protein production was dependent on the addition of cytokines such as IFN-{gamma} (44). The HOG cell line was found to be complement resistant even after inhibition of CD59 and DAF, but the role of factor H in the resistance was not studied. In our study, we demonstrate that H2 cells produce factor H and FHL-1 constitutively in the absence of cytokine stimulation and that both proteins play a role in complement resistance. Because FHL-1 shares the complement-regulating functions with factor H and contains domains relevant for cell attachment (28, 29), it can be suggested that FHL-1 also protects H2 cells against complement activation. The higher relative proportion of FHL-1, as compared with factor H, in the H2 cell growth supernatant suggests that FHL-1 may even be more important than factor H.

The resistance of autologous cells against complement-mediated damage is mainly mediated by the membrane-bound glycoproteins MCP (CD46), DAF (CD55), and protectin (CD59) (1). We therefore examined the expression of these complement regulators on H2 cells (Fig. 1Go) and the effect of their neutralization on complement sensitivity. In most cases, nucleated cells can be sensitized to complement lysis by neutralizing CD59. Upon neutralization of CD59, 40% of U251 glioma cells were killed in a standard lysis assay (Fig. 2GoA). The level of lysis with the endothelial-like EA.hy 926 cells was 70%, whereas H2 cells remained extremely resistant to lysis after neutralization of CD59 (Fig. 2GoA). Moreover, H2 glioblastoma cells were protected even when all of the three complement regulators, CD46, CD55, and CD59, were neutralized (Fig. 2GoB). This resistance is unusual, because all other cell lines we have tested to date were killed at least to some degree after blocking of one or more of the three regulators (25, 26, 45). The fact that treatment of H2 cells with PIPLC, which removed 60% and 69% of GPI-anchored CD55 and CD59, respectively, from the cell membranes, had no effect on lysis (Fig. 3GoA) indicated that these inhibitors did not account for the exceptional complement resistance of H2 cells. Furthermore, neither heparan sulfate nor cysteine proteases, such as p41 (46), seemed to be responsible for the resistance of H2 cells.

Treatment of H2 cells with trypsin, however, strongly increased complement-mediated lysis of H2 cells (Fig. 3GoC), showing that cell surface proteins and structures attached to them are responsible for the resistance. Earlier studies (15, 17, 18) have shown that sialic acid strongly contributes to factor H-mediated resistance to complement. We therefore treated H2 cells with neuraminidase and found a considerable increase in complement-mediated lysis (Fig. 3GoB). This observation further supported our conclusion that H2 cells utilize factor H for their protection. The involvement of factor H was confirmed by the demonstration that following Ab and serum treatment, conversion of C3b to iC3b was more efficiently promoted by H2 cells and that less C5b-9 deposition was detectable on H2 cells than on U251 and EA.hy 926 cells (Fig. 4Go). This difference could not be explained by MCP because no significant difference in MCP expression between the cell lines was observed (Fig. 5Go) (24).

Because none of the known mechanisms appeared to explain the resistance of H2 cells, we considered the possibility that H2 cells themselves produced other regulators, such as factor H. Direct evidence for the expression of factor H mRNA and protein production by the H2 cells was obtained from RT-PCR, immunoblotting, and ELISA analyses. By RT-PCR, we found that the H2 cells, but none of the other cell lines, U251, EA.hy 926, T47D (37), Paju (39), examined, strongly expressed not only factor H, but the FHL-1 mRNA (Fig. 5GoA). Immunoblotting (Fig. 5GoB) and ELISA analyses showed that H2 cells secreted factor H and FHL-1 into the growth medium. Interestingly, both at the RNA and protein level, FHL-1 production exceeded that of factor H. This is different from human serum, in which FHL-1 concentration is 10–50 times lower than that of factor H. Subsequent to this study, we have observed that another complement-resistant glioma cell line (U138) secretes both factor H and FHL-1, whereas a complement-sensitive glioma cell line (U87) secreted no factor H and only a trace amount of FHL-1 under identical conditions. Thus, it appears that the expression and secretion of soluble complement regulators could be a more general phenomenon in providing complement resistance to tumors.

Having demonstrated that H2 cells secreted factor H and FHL-1, we analyzed by FACS whether H2 cells were able to recruit factor H from the fluid phase to their cell surfaces (Fig. 6Go). Earlier studies have shown that factor H can bind to polymorphonuclear leukocytes, U937 monocytic cells, and Raji B lymphoblastoid cells (47, 48, 49). As one of these reports had demonstrated that factor H bound to the iC3b receptor (CR3, CD11b/CD18) on polymorphonuclear leukocytes (48), we examined whether H2 cells expressed this protein, but no expression was found (data not shown). Binding studies using radiolabeled factor H showed that factor H bound to H2 cells in a saturable manner (Fig. 6GoB) and binding could be inhibited by unlabeled factor H (Fig. 6GoC). Scatchard analysis, however, indicated that factor H binding was heterogenous in nature. Nonlinearity of the binding curve could be due to varying affinities and/or different numbers of receptors for factor H on the cell surface. Further studies are required to determine the nature of a possible high affinity receptor on H2 cells. The low affinity binding of factor H, and possibly also of FHL-1, to the H2 cell surface could be mediated by sialic acids. This suggestion is supported by the observation that after removal of surface-associated sialic acid, the H2 cells became more sensitive to complement-mediated killing (Fig. 3GoB). Under physiological conditions, the binding of factor H usually occurs in the context of initial C3b deposition. Acquisition of factor H to such surface efficiently stops complement activation.

To study the functional significance of factor H and FHL-1 on the complement resistance of H2 cells, we used specific mAb to inactivate the two proteins. Because factor H has multiple binding sites for C3b (34, 50), a polyclonal Ab or a combination of anti-factor H mAb is needed for its complete neutralization. Using a combination of two anti-factor H mAbs (131X and OX24) together with the YTH53.1 mAb in a cytotoxicity assay lysis of H2 cells increased 5-fold. This efficient sensitization of H2 cells to complement-mediated killing suggested a protective role for factor H and FHL-1. However, as the assay (see Materials and Methods) lasts ~1 h and the cells are thoroughly washed before treatment with Abs and NHS, it cannot be excluded that factor H is acquired from serum. Thus, it is evident that H2 cells not only synthesize and secrete factor H and FHL-1, but they can also recruit at least factor H from the fluid phase (Fig. 6Go). As a consequence, H2 cells can utilize both endogenously synthesized and fluid-phase factor H and FHL-1 from plasma to efficiently regulate complement activation and prevent terminal complement component deposition on their cell membranes. This effect is probably most benefical for the tumors in their local environment in the CNS, in which the tumor cells persist for prolonged periods and possibly create a complement-free zone around them. In conclusion, our results demonstrate a regulatory role for factor H and FHL-1 in the complement resistance of H2 glioblastoma cells, and thus suggest a novel mechanism whereby malignant cells can escape attack by the complement system.


   Acknowledgments
 
We thank Drs. J. Jääskeläinen, A. Sankila, and T. Timonen for providing the H2 and U251 glioma cell lines, and Dr. V. Koistinen for help in protein purification.


   Footnotes
 
1 This work was supported by grants from the Academy of Finland, the Sigrid Jusélius Foundation, the University of Helsinki, the Helsinki University Hospital Research Funds, the Deutsche Forschungsgemeinschaft (Zi 432/5-1), and the Deutscher Akademischer Austauschdienst (DAAD). Back

2 Address correspondence and reprint requests to Dr. Seppo Meri, Department of Bacteriology and Immunology, Haartman Institute, P.O. Box 21, FIN-00014 Helsinki, Finland. Back

3 Abbreviations used in this paper: CR1, complement receptor 1; DAF, decay-accelerating factor; FHL-1, factor H-like protein 1; MCP, membrane cofactor protein; NHS, normal human serum; PIPLC, phosphoinositol-specific phospholipase C; SCR, short consensus repeat. Back

Received for publication August 9, 1999. Accepted for publication March 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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