Immunogenetics 31: 145-151, 1990 lmm o- geneO s

© Springer-Verlag 1990

Synthesis of complement component C5 by human B and T lymphoblastoid cell lines

William Reed 1, Robert A.S. Roubey 1, Juanita G. Dalzeli 1, Barbara M. Matteucci 1'*, Barry L. Myones l't, Stephen W. Hunt, HI 1, William P. Kolb 2, and Gordon D. Ross I

1 The Division of Rheumatology and Immunology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 2 Quidel, Inc., San Diego, CA 92121, USA

Received September 4, 1989; revised version received November 20, 1989

Abstract. Human B and T lymphoblastoid cell lines were shown to synthesize C5. C5 synthesis was quantitated with an enzyme-linked immunosorbent assay (ELISA) that utilized a pool of C5-specific monoclonal antibodies (mAbs). Some level of C5 synthesis was detected in all eight of the B and T cell lines examined. In three of the cell lines, C5 was detected in both culture supernatants and whole cell detergent lysates, whereas in the other five cell lines, C5 was detected only in the cell lysates. Lym- phoblastoid cells with both distributions of C5 were shown to synthesize a messenger RNA that was similar in size to the C5 mRNA expressed by the HepG2 hepatoma cell line. Estimates of the concentration of the C5 transcript in poly(A) ÷ RNA from lymphoblastoid and HepG2 cells suggested that C5 mRNA levels in the lymphoblastoid cell lines were comparable and about one-tenth of the levels in HepG2 cells. Lymphoblastoid C5, isolated by im- munoaffinity chromatography from the supernatants of 35S-labeled cultures, had the same subunit composition as plasma-derived C5, but had an e¢ subunit of slightly smaller relative mass.

Introduction

C5 is a component of the terminal pathway of complement (C) which participates in both the inflammatory and cytolytic processes that follow the activation of C by either the classical or alternative pathway. When isolated from

* Present address: Division of Rheumatology, Hahnemann University Medical School, Philadelphia, Pennsylvania, USA.

~ Present address: Department of Pediatrics, Children's Hospital Medical Center, Northwestern University, Chicago, Illinois, USA.

Address correspondence and offprint requests to: Dr. William Reed, Division of Rheumatology and Immunology CB # 7280, 932 F.L.O.B, 23 l-H, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7280, USA.

plasma, C5 is a glycosylated heterodimer composed of disulfide-linked ot (M r 115000) and t3 (Mr 75000) subunits (Nilsson et al. 1972; Tack et al. 1979). The subunits are encoded by a single messenger RNA (mRNA) that directs the synthesis of pro-C5, an M r 190000 precursor with a/3-a organization (Ooi and Colten 1979; Patel and Minta 1979; Lundwall et al. 1985; Wetsel et al. 1988). Pro-C5 is post-translationally processed into the mature heterodimer prior to secretion. Secreted C5 is ac- tivated upon the specific proteolytic excision of residues 1-74 from the o~ subunit. The release of this small activa- tion peptide, C5a (M r 9000), triggers several types of in- flammatory reactions via specific cellular receptors (Chenoweth 1986; Janatova 1988). The larger C5 frag- ment, C5b (M r 181 000), takes part in cytolytic events by initiating the assembly of the C5b-9 complex, the mem- brane attack complex which mediates irreversible mem- brane damage (reviewed by Miilter-Eberhard 1988).

Plasma contains small amounts of C5 (ca. 75 ~tg/ml), most of which is thought to be synthesized by hepatocytes (Colten 1976; Geng et al. 1986), although it is certain that there are other, local sources of C5. Macrophages and specific types of epithelial cells and fibroblasts have been shown to synthesize C5 (Ooi and Colten 1979; Strunk et al. 1988; Rothman et al. 1989), and monocytes may as well (Sundsmo and G6tze 1981; Hetland et al. 1986a, b). Expression in macrophages may vary with species, tissue of origin, and differentiation state. Surprisingly, mono- cytes cultured in vitro for 3 days express C5 immunoreac- tivity associated with their surface membrane which has been implicated in macrophage spreading reactions induc- ed by the Bb fragment of factor B (Sundsmo and G6tze 1981).

Lymphocytes may also synthesize C5 (Sundsmo et al. 1979), although an explicit demonstration of this has been precluded by the presence of monocytes in preparations of peripheral blood lymphocytes (PBLs). A possible role for C5 in lymphocyte responses has been implied by the

146 W. Reed et al.: Lymphocyte synthesis of C5

observation that anti-C5 Fab' fragments inhibit mitogenic responses initiated with phytohemagglutinin or a mixed lymphocyte reaction (Sundsmo 1983). In addition, C5a can enhance both antigen-specific and non-specific an- tibody responses in vitro (Morgan et al. 1984) and can trigger or potentiate lymphocyte proliferative responses (Sundsmo 1983; Morgan et al. 1984). Taken together, these observations raise the possibility that locally syn- thesized C5, especially when lymphocyte-derived, may perform functions not directly involved with the inflam- matory and cytolytic functions normally associated with C5. In this study, established lines of B and T lym- phoblastoid cells that are totally free of contaminating monocytes were examined for C5 synthesis.

Materials and methods

Cell Lines. The B-lymphoblastoid lines Raji, WIL 2, U698M, and Matthews, the T lymphoblastoid lines MOLT-3, MOLT-4, HSB, and 8402, and the monocytoid line U937 were obtained from the Tissue Culture Facility of the Lineberger Cancer Research Center of the University of North Carolina at Chapel Hill. Each of the lines was main- tained in RPMI-1640 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 5 mM sodium pyruvate, 10 mMhydroxyethyl- piperazine-ethanesulfonic acid (HEPES), and antibiotics. The hepatoma cell line HepG2 (Knowles et al. 1980; Morris et al. 1982) was maintained in Eagle's minimal essential medium with Earle's balanced salts, 10% fetal calf serum (FCS), 5 rnM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, and antibiotics.

Antibodies. Goat antiserum to C5 was generated by intramuscular im- munization with five biweekly 200 gg injections of purified C5 (Lambris et al. 1980) emulsified in Freund's adjuvant, using the complete adjuvant for the first immunization and incomplete adjuvant for booster injec- tions. The antiserum was specific for C5 among human serum proteins when analyzed by immunodiffusion and immunoelectrophoresis. No cross-reactivity with proteins present in FBS was detected by im- munodiffusion. The IgG fraction was isolated by chromatography on DEAE-Sephacel (Pharmacia Fine Chemicals, Piscataway, New Jersey) in 20 mM sodium phosphate buffer pH 7.0, and further purified by a second round of diethylaminoethanol chromatography using high perfor- mance liquid chromatography (Klapper et al. 1986; Myones et al. 1988). For enzyme-linked immunosorbent assay (ELISA), 3 mg of the IgG anti- C5 was biotinylated (Bayer and Wilcheck 1980). For immunoaffinity isolation of C5, IgG anti-C5 was coupled to cyanogen bromide-activated Sepharose (Pharmacia) at a ratio of 10 mg IgG/ml gel. Thirty-seven monoclonal antibodies (mAbs) to C5 were generated by standard techni- ques using immune splenocytes from Balb/c mice and NS-1 myeloma fusion partners (Goding 1980).

ELISAsfor C5. A sandwich assay was used in which 96-well Immulon micro-ELISA plates (Dynatech, Alexandria, Virginia) were coated with 100 ~tl/well goat IgG anti-C5 (100 Ixg/ml), blocked with 0.2 M sodium borate, 75 mM sodium chloride, pH 8.4 (borate buffered saline; BBS) containing 0.5 % bovine serum albumin and 0.4 % polyoxyethylene sor- bitan mono-oleate (TWEEN 80; Sigma, St. Louis, Missouri), and then incubated with the sample containing C5. In the polyclonal ELISA, C5 bound by the first antibody was detected by addition of 100 Ixl biotinylated goat IgG anti-C5 (10 Ixg/ml). In the monoclonal assay, 100 gl mouse C5-specific mAb (tissue culture supernatants diluted 1 : 100) followed by 100 ~tl biotinylated goat anti-mouse IgG (diluted 1 : 500, Sigma) was used. Thirty-seven C5-specific mAbs were evaluated for

their ability to detect low concentrations of purified C5 in the ELISA. A mixture of three particular mAbs was identified that allowed a slightly higher sensitivity to low levels of C5 than any of the individual mAbs in the mixture. Both the polyclonal and monoclonal assays were developed by incubations with 100 gl/well avidin-alkaline phosphatase (diluted 1 : 1000; Zymed Laboratories, South San Francisco, Califor- nia), followed by 100 gl/well p-nitrophenylphosphate substrate (1 mg/ml, Sigma). Wells were washed with BBS four times between each step using an automatic microplate washer (Skatron, Lier, Nor- way). All incubations were performed at room temperature. For each assay, negative controls were included in which the initial coating with anti-C5 was omitted. Dilutions of immunoaffinity-purified C5 (Wetsel et al. 1980; Quidel, San Diego, California) were tested in parallel with cell culture samples to establish assay sensitivity and generate a standard curve for the calculation of sample C5 concentrations.

Immunoprecipitation and fluorography. Metabolic 35S-labeling of cell proteins was accomplished by overnight cultivation of 5 × 106 cells in 100 ml methionine/cysteine-free RPMI-1640 (Selectamine TM kit, Gib- co, Grand Island, New York), 5% FCS, 5 mM sodium pyruvate, 10 mM HEPES, antibiotics, and 5 mCi of a mixture of 70% 35S- methionine and 20% 35S-cysteine (Trans 35S-labelTM, ICN Radio- chemicals, Irvine, California). Following sedimentation, medium and cell pellet were analyzed separately for the presence of 35S-labeled C5. Spent medium was dialyzed against phosphate buffered saline (PBS) at 4 °C using SPECTRA/POR type 6 dialysis tubing (Fisher Scientific, Pittsburgh, Pennsylvania). The cell pellet was resuspended in 2 ml ice- cold lysis buffer [50 mM Tris-HC1 pH 7.4, 100 mM KC1, 10 mM ethylenediaminetetraacetate (EDTA), 0.1 mM p-amidinophenylmethyl- sulfonyl fluoride (Calbiochem-Behring, La Jolla, California), 0.25 mM p-nitrophenyl p'-guanidino benzoate, 1 mg/ml soybean trypsin inhibitor, 1 mg/ml aprotinin, 20 ~tM pepstatin A, 100 mM ~-amino caproic acid, and 75 mMbenzamidine (Sigma)] and solubilized on ice with rapid stirr- ing by addition of Triton X-100 and deoxycholate (Sigma), each at a final concentration of 0.5 %. Insoluble cellular debris was removed by centrifugation at 40000 g for 30 min at 4 °C. Dialyzed culture medium and solubilized cell pellet extract were run on serial columns of a) 10 ml of human Cohn Fraction II IgG-Sepharose and b) 1 ml of anti- C5-Sepharose. Following sample application, the columns were washed with 400 ml equilibration buffer [3.5 mM veronal buffered sodium chloride (2.5-3.5 mS at 22 °C), pH 7.3] and separated. They were then washed sequentially with 200 ml of each of the following: veronal buf- fered saline (13.8 mS), veronal buffered saline/0.1% Nonidet P40 (NP-40; Particle Data Laboratories, Elmhurst, Illinois), and 10 mM phosphate buffered 0.6 M NaC1/0.1% NP-40, pH 7.5 (55 mS). Bound proteins were eluted with 50 ml 4 M guanidine hydrochloride. Elnates were dialyzed extensively against PBS to remove NP-40 and concen- trated with an Amicon YM-10 membrane (Amicon, Danvers, Massachusetts) to - 5 ml. Remaining NP-40 was removed by incubation with 600 mg Bit-beads SM-2 (Bit Rad Laboratories, Richmond, California) on a rocking platform for 1.5 h at 4 °C. Bit-beads were removed by filtration and washed once with 30 ml PBS. Eluates were finally concentrated to approximately 50 ~tl with YM-10 membranes followed by Centricon- 10 filtration units (Amicon). Samples were boiled for 5 min with sodium dodecyl sulfate (SDS) dithiothreitol, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (5 % gel; Laemmli 1970). Purified plasma C5 and relative-mass markers (Bit Rad) were run in adjacent lanes. Gels were silver stained, incubated 30 min in Enlightning (New England Nuclear, Boston, Massachusetts), dried, and exposed to Kodak X-OMAT AR fdm (Kodak, Rochester, New York), for 1-14 days at - 8 0 ° C .

RNA isolation. Total RNA was isolated from 4 M guanidium isothio- cyanate (Sigma) lysates of washed cells by sedimentation through a cushion of 5.7 M cesium chloride (International Biotechnologies, New Haven, Connecticut; Davis et al. 1986). RNA enriched in poly(A) +

W. Reed et al.: Lymphocyte synthesis of C5 147

transcripts was isolated from 2 % SDS (BRL, Galthersberg, Maryland) lysates of washed cell pellets by affinity chromatography using oligo(dT)-cellulose (type 7 from Pharmacia) as described (Badley et al. 1988). In some cases, a second round of affinity chromatography was performed to increase poly(A) + RNA purity.

Preparation of radiolabeled probes. The 0.95 kilobase (kb) Pst I-Hind III fragment of a partial human C5 cDNA (Lundwall et al. 1985) was subcloned into the phagemid pBluescript KS + (Stratagene, La Jolla, California) where it served as template for in vitro transcription of single- stranded, 32P-labeled RNA probes. C5 mRNA sense and anti-sense RNA probes were transcribed from linearized plasmid (digested with Hind III or Barn HI, respectively) in the presence of [32p]UTP (ICN Radiochemicals) using an RNA transcription kit according to the sup- plier's instructions (Strategene). Size analysis of the probe by elec- trophoresis through 5 % polyacrylamide, 7 M urea, denaturing gels con- firmed that over 95 % of the synthesized probe correspond to full-length transcripts of the template cDNA.

Alkaline northern blot analysP~. RNA transcripts were separated accord- ing to size by overnight electrophoresis through 1% agarose for- maldehyde gels (Davis et al. 1986), and transferred to Zeta-Probe nylon membranes (BioRad) by capillary action using 5 mM NaOH as transfer buffer. Membranes were prehybridized, hybridized, and washed as described (Vrati et al. 1987), except that 0.5 mg/ml denatured yeast RNA (Sigma) was included in the hybridization buffer and the filters were washed with 0.5 mg/ml RNase A (Sigma).

Solution hybridization/RNase protection assay. Total or poly(A) + RNA was combined with an excess of 32P-labeled sense or anti-sense RNA probe in hybridization buffer (0.86 M NaC1, 6.7 mM EDTA, 0.2 % SDS, 7% ethanol, 30 mM Tris-HC1, pH 7.5), overlaid with paraffin oil, and incubated overnight at 80 °C. Unhybridized probe was digested with RNase A and T1 (Sigma) and the RNase-resistant RNA-RNA hybrids were precipitated by the addition of carrier DNA and trichloroacetic acid. The precipitate was collected on nitrocellulose or glass fiber filters, washed, and counted by scintillation spec- trophotometry.

Results

Detection o f C5 in B and T lymphoblastoid cell lines. C5 was detected in culture supernatants from certain B (WIL 2, U698M) and T (MOLT-4) lymphoblastoid lines by an ELISA that used a goat polyclonal anti-C5 for both antigen capture and detection (Table 1). Tests with purified plasma C5 indicated that this polyclonal ELISA was sen- sitive to levels of C5 as low as 0.1 gg/ml. The presence of C5 in the culture supernatants was confirmed using a second ELISA that employed a mixture of three C5-specific mAbs. The monoclonal and polyclonal ELISAs had approximately the same sensitivity to low concentrations of C5.

Despite the failure to detect C5 in the culture super- natants of some of the cell lines, C5 was detected in detergent-solubilized cells from each of the B and T cell lines using both the polyclonal and monoclonal ELISA. Thus, the lymphoblastoid cell lines were of two kinds, those for which C5 was detected in both culture super- natants and whole cell detergent lysates, and those for

Table 1. ELISA for C5 in culture supernatants and detergent-solubilized cell pellets from B and T lymphoblastoid, monocytoid, and hepatoma cell tines.

Cell line C u l t u r e Detergent-solubilized supernatant (ng/ml*) cell pellet*

B cells

WIL 2 25.0 5.0 U698M 0.10 1.3 Raji < 0.10 4.0 Matthews < 0.10 100.0

T cells

MOLT-4 2.0-10.0 5.0 HSB <0.10 1.8 8402 < 0.10 0.5 MOLT-3 < 0.10 39.0

Monocytoid Line

U937 < 0.10 < 0.10

Hepatoma Line

HepG2 1000 46

* A washed cell pellet of 1 × 10 7 cells was solubilized with a mixture of 0.5% Triton X-100/0.5% deoxycholate (see Materials and methods).

t Values in nanograms/ml are the mean of 3-4 assays performed with both the polyclonal and monoclonal ELISA in which the second detect- ing antibody was either goat anti-C5-biotin (polyclonal ELISA) or mouse monoclonal anti-C5 followed by goat anti-mouse IgG-biotin (monoclonal ELISA).

which C5 was detected in cell lysates alone. Solubilization of C5 from whole cells and detection by ELISA required a mixture of a non-ionic detergent such as Triton X-100 and the weakly ionic detergent deoxycholate. Little or no C5 was detected after solubilization with NP-40 or Triton X-100 alone. Similar results have been reported previous- ly in studies of C5 synthesis by mouse macrophages (Ooi and Colten 1979).

Tests of HepG2 cells, a human hepatoma cell line that is known to synthesize C5 (Morris et al. 1982), confirmed the presence of relatively high levels of C5 in both culture supernatants and solubilized cells. The amount of C5 detected per cell in most of the lymphoblastoid cell lines was one-tenth or less of the amount of C5 detected per HepG2 cell (Table 1). The MOLT-3 and Matthews cell lines were exceptions; they yielded comparable or greater levels of C5 per cell compared to HepG2, in spite of the fact that C5 was undetectable in their culture supernatants.

Isolation o f metabolically radiolabeled C5. The specifici- ty of the polyclonal anti-C5 was confirmed by im- munoprecipitation and western blot analysis. Anti- C5-Sepharose immunoprecipitated C5 from normal serum as determined by comparing the immunoprecipitate to a plasma C5 standard (Cytotech) by reducing SDS-

148 w. Reed et al.: Lymphocyte synthesis of C5

PAGE (not shown). The anti-C5 detected the plasma C5 standard on western blots and anti-C5-Sepharose im- munoprecipitated a high relative mass polypeptide from HepG2 cells with a mobility appropriate for pro-C5 (not shown), in agreement with previous studies (Morris et al. 1982).

WIL 2 B-type lymphoblastoid cells were cultured overnight in medium containing 35S-labeled cysteine and methionine. The culture supernatant was examined for radiolabeled C5 by immunoaffinity chromatography over goat polyclonal anti-C5-Sepharose followed by SDS- PAGE, and fluorography. The eluate from anti- C5-Sepharose contained two major 35S-labeled polypep- tides (Fig. 1, lane 3). The smaller polypeptide co- migrated with the silver nitrate-stained/3 subunit of C5 purified from plasma (Fig. 1, lane 4). However, the larger polypeptide was slightly smaller than the ot subunit of C5. In addition to the two major radiolabeled polypeptides, a minor 35S-labeled species migrating with a mobility ap- propriate for pro-C5 (-190 000) is also present in the anti- C5 immunoaffinity column eluates (Fig. 1, lane 3). These three radiolabeled polypeptides were not detected in elutes

Fig. 1. Isolation of radiolabeled C5 from the culture supernatant of WIL 2 B-type lymphoblastoid ceils. Two major radiolabeled polypeptides were isolated by anti-C5 immunoaffinity chromatography of overnight culture supernatants from WIL 2 cells grown in the presence of 35S-labeled cysteine and methionine. Reducing SDS-PAGE and fluorographic analysis of the immunoaffinity column eluate is shown in lane 3. The smaller polypeptide co-migrates with the/~ subunit of serum-derived C5, while the larger one migrates just below the c~ subunit of C5 (lane 4). A much weaker polypeptide that migrates just below 200 000 may be pro-C5. Relative mass markers are shown in lanes 1 and 2,

of the Cohn Fraction n IgG-Sepharose column control (not shown).

The reason for the reduced size of the o~ subunit is unclear. Cleavage by unknown proteases in the media is one possibility. However, incubation of 125I-labeled plasma C5 at 37 °C in media with or without WIL 2 cells for 3 days did not produce a reduction in ot subunit size (not shown). This does not exclude the possibility of pro- teolytic activity in the particular batch of FCS used for the biosynthetic labeling experiments. We have previous- ly noted cleavage of Raji cell CR2 in spent culture media (Myones and Ross 1987), and this was later shown to be due to proteases contained in the FCS media supplement (B. Myones and T. Schulz, unpublished observation). Alternatively, the smaller ~ subunit may result from dif- ferent post-translational processing as compared to plasma C5, such that the ot subunit lymphocyte C5 con- tains less protein or carbohydrate.

Detection and quantitation of C5 messenger RNA. The C5 mRNA from HepG2 cells, originally described by Lund- wall and co-workers (1985), was used as a standard for the detection of C5 mRNA in the lymphoblastoid cell lines. Four of the cell lines characterized by ELISA were examined by alkaline northern blot analysis. All four cell lines expressed C5 mRNA as judged by the presence of an RNA ,species that co-migrated with C5 mRNA from HepG2 ceils (Fig. 2B). In addition, two of the cell lines expressed a smaller, minor transcript that was also present in HepG2 cells; the identity of this minor transcript is unknown.

The concentration of C5 mRNA in HepG2 and lymphoblastoid cell RNA was estimated by a solution hybridization/RNase protection assay that employed the same probe used in northern blot hybridizations. An ex- ample of this assay is shown in Figure 3. There was a linear relationship between the amount of lymphoblastoid poly(A) + RNA added and the amount of C5 anti-sense RNA probe that was protected from digestion with RNase A and T1 (solid circles, Fig. 3). Total RNA from HepG2 cells also protected the C5 anti-sense probe (solid squares, Fig. 3). In contrast, neither lymphoblastoid nor HepG2 RNA protected the corresponding C5 sense RNA probe (open symbols, Fig. 3). The slope of these curves is an estimate of C5 mRNA concentration.

C5 mRNA concentrations in poly(A) ÷ RNA from the four lymphoblastoid cell lines and HepG2 cells are shown in Table 2. These estimates reflect the relative concentra- tions of C5 message among the five cell fines. The concen- trations of C5 mRNA in the four lymphoblastoid cells were all similar and consistently about one-tenth of the concentration in HepG2 cells. Three of the cell lines syn- thesize about one-tenth as much C5 protein as HepG2 determined by ELISA of detergent-solubifized cells (Table 1). The Matthews line is an exception to this cor-

W. Reed et al.: Lymphocyte synthesis of C5

A

Pstl Hindlll

I . . . . . . . . . .

B

149

Table 2. C5 mRNA concentration in poly(A) + RNA from HepG2 and lymphoblastoid cell lines. Quantitation by solution hybridization/RNase protection assay*.

Cell line RNase-resistant probe C5 mRNA detected cpm/~tg pg/pg+

HepG2 747 29.4 Matthews 52 2.0 MOLT-4 59 2.3 Raji 62 2.4 WlL 2 66 2.6

* All RNA fractions were ca. 50% poly(A) ÷ RNA from a single round of affinity chromatography over oligo(dT)-cellulose (see Materials and methods for details).

+ Calculated from the RNase-resistant probe data based upon a probe specific activity of 140 cpm/pg and assuming a 944 nucleotide pro- tected fragment and a 5.2 kb C5 mRNA.

Fig. 2 A, B. Identification of C5 messenger RNA in lymphoblastoid cell lines. The structure of C5 mRNA is shown diagrammatically in A. The open box represents the open-reading frame of the mRNA with its /3-a subunit-encoding organization. The C5 anti-sense probe hybridizes to a 944 nucleotide sequence that crosses the t3-a junction of the message (black bar). B An alkaline northern blot of HepG2 (H), Matthews (M), MOLT-4 (Mo), Raji (R), and WIL 2 (W) RNA was hybridized with the C5 anti-sense RNA probe revealing a lymphoblastoid cell RNA species that co-migrates with the 5.2 kb C5 mRNA synthesized by HepG2 cells. Lane H, 15 ~tg HepG2 total RNA; all other lanes contain 20 pg of poly(A) + RNA from the indicated lymphoblastoid cell line,

7,000 ~ H e "~ 6,000

"~" pG2 ~ "°°°I / "~ u~ 3,000

~ 2000 MOLT-4

I'~ 0 5 10 15 20 25

RNA ( micrograms )

Fig. 3. Quantitation of C5 mRNA recovery from HepG2 and MOLT-4 ceils. The same C5 anti-sense probe used for northern blot hybridizations was used in a solution hybridization/RNase protection assay (see Materials and methods) to estimate the concentration of C5 mRNA in RNA prepared from HepG2 and MOLT-4 cells. Both total HepG2 RNA and MOLT-4 poly(A) + RNA protect the C5 anti-sense RNA probe from digestion with RNase A and T1, but fail to protect the correspond- ing C5 sense RNA probe from digestion (open symbols).

respondence between the concentrations of C5 messenger and protein. Matthews cells exhibited the highest C5 pro- tein concentration, but did not show a higher concentra- tion of C5 m R N A compared to the other lymphoblastoid cell lines.

F rom assays of HepG2 total RNA, we have estimated that C5 m R N A is present in the range of 50 copies per cell, making it a rare message. C5 mRNA was not detected in lymphoblastoid cell total RNA by our assay, confirming that its concentration in these cell lines is significantly lower than that in HepG2 cells. Based on the results in Table 2, we estimate that C5 m R N A is present in lym- phoblastoid ceils in less than five copies per cell.

Discussion

Previous studies have shown that hepatocytes, macrophages, and some types of epithelial cells and fibroblasts synthesize C5 (Patel and Minta 1979; Ooi and Colten 1979; Hetland et al. 1986a; Geng et al. 1986; Strunk et al. 1988; Rothman et al. 1989). Past reports that examined lymphocytes for C5 synthesis have had disparate findings. Sundsmo and colleagues (1979) detected C5 synthesis in preparations of PBLs but could not exclude the possibil i ty that this C5 was derived from contaminating monocytes (Sundsmo et al. 1979; Sundsmo 1982). Attempts to characterize the structure o f this lym- phocyte C5 were also inconclusive because the biosyn- thetically labeled protein immunoprecipi tated by anti-C5 consisted o f several protein bands on SDS-PAGE gels that did not clearly correspond to the mobili t ies of C5 ~ and /3 chains (Sundsmo 1982). Another study from that same per iod failed to detect lymphocyte C5 synthesis (Kolb and Kolb 1982). The current investigation demonstrated that established lymphoblastoid cell lines o f B- and T-cell origin synthesize small amounts of C5 m R N A and that

150 W. Reed et al.: Lymphocyte synthesis of C5

they synthesize and, at least in some cases, secrete C5 that is immunologically and biochemically related to C5 isolated from plasma. However, unstimulated normal lymphocytes were not examined and it remains possible that C5 synthesis may require Epstein-Barr virus transfor- mation or lymphocyte activation.

The lymphoblastoid C5 that was isolated from the spent media of the WlL 2 cell line had the same subunit composition as plasma C5, but had an a subunit of slightly smaller relative mass. This is surprising, because the lym- phoblastoid C5 mRNA is similar in size to C5 message from HepG2 cells, which is believed to be identical to the message that encodes plasma C5 (Lundwall et al. 1985). We cannot rule out the possibility that there are subtle dif- ferences between lymphoblastoid and HepG2 C5 mRNA that are not detectable by northern blot analysis or that the different cell types process the same translation pro- duct differently. Proteolytic cleavage of the secreted C5 also may have occurred. Although incubation of ~25I-labeled plasma C5 with WlL 2 cells did not result in a chain cleavage, additional proteolytic activity may have been present in the particular batch of FCS used for the biosynthetic labeling experiments. A number of common proteases cleave the C5 ot subunit, yielding a fragment with mobility similar to that seen in Figure 1 (Wetsel and Kolb 1983). Microsequence analysis of the N-terminal of the C5 a subunit will most likely be required to discriminate between protein or carbohydrate differences that contribute to the smaller a subunit size of lym- phoblastoid cell-secreted C5.

The Raji cells are similar to MOLT-4 and WlL 2 cells in all respects except that there is no detectable C5 in the culture supernatant. This may be due to greater proteolytic activity in the Raji supernatants. The Matthews cell line was an even greater exception. It did not show the cor- respondence between C5 mRNA and protein synthesis, and although it synthesized the greatest quantities of C5 detectable by ELISA, none was detectable in the culture supernatant. Based on these anomalies, we cannot rule out the possibility that lymphoblastoid cells synthesize alter- native forms of C5 that are preferentially membrane- associated.

The function of C5 expressed by lymphocytes or macrophages is uncertain, but it appears untikely that C5 derived from lymphocytes or macrophages makes up a significant proportion of the total plasma pool of C5 (Geng et al. 1986). Initial studies suggest that many of the lym- phoblastoid lines resemble monocyte-derived macro- phages (Sundsmo and G6tze 1981), in that they express C5 immunoreactivity associated with the membrane sur- face (Roubey et ai. 1989). Experiments with macrophages had suggested that membrane C5 might be involved in factor B (Bb)-mediated activation events. Other more recent reports have shown that macrophages can utilize intrinsic secreted C proteins to opsonize particles such as

agarose beads or IgM antibody-coated erythrocytes for phagocytosis (Hetland and Eskeland 1986, 1987). However, C5 is not required for C activation or opsoniza- tion of particles with fixed C3. The studies of factor B- induced monocyte spreading reported by Sundsmo and G6tze (1981) had suggested that monocyte surface C5 might serve as a substrate for the proteolytic activity of Bb. Fab' anti-C5 inhibited Bb-induced monocyte spreading, whereas spreading was induced by F(ab') 2 an- ti-C5. Although it was also reported that Bb induced lym- phocyte blastogenesis and that Bb-induced responses were inhibited by Fab' anti-C5 (Sundsmo et al. 1982; Sundsmo 1983), others have recently reported that Bb-induced lym- phocyte proliferation did not require an active proteolytic site in Bb and appeared to be due to the binding of Bb to the B-cell receptor for high relative mass B-cell growth factor (Peters et al. 1988).

Future studies will focus on investigation of C5 syn- thesis and membrane surface expression by freshly isolated lymphocytes. Among the 37 mAbs to C5 examin- ed in the present study are several mAbs that have the ability to inhibit the hemolytic activity of serum C5 as well as to bind to epitopes of C5 expressed on the cell surface of B and T cell lines. Thus, these mAbs should be ideal for future investigations of the possible functions of lym- phocyte C5.

Acknowledgments. This work was supported by a research grant from the American Cancer Society (IM-308F). Dr. Myones was a Special Postdoctoral Fellow of the Leukemia Society of America while some of this work was being performed. The authors are grateful to Dr. Brian F. Tack, Research Institute of Scripps Clinic, La Jolla, California, for the generous gift of the C5 cDNA probe J-16.

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