Expression of H-ficolin/Hakata antigen, mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 by human glioma cell line T98G

Mikio Kuraya1, Misao Matsushita1, Yuichi Endo1, Steffen Thiel2 and Teizo Fujita1

1 Department of Biochemistry, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1295, Japan 2 Department of Medical Microbiology and Immunology, University of Aarhus, DK 8000 Aarhus, Denmark

Correspondence to: M. Kuraya; E-mail: mkuraya{at}fmu.ac.jp
Transmitting editor: S. Izui


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense and it requires MBL-associated serine proteases (MASP) for activation of the lectin complement pathway (LCP). Recently we reported that human ficolins, L-ficolin/P35 and H-ficolin/Hakata antigen, as well as MBL activate the LCP in association with MASP. We investigated in vitro expression of complements of the lectin complement pathway in several cell lines. Out of 17 cell lines tested using RT-PCR, a human glioma cell line, T98G, expressed high levels of H-ficolin/Hakata antigen, MASP1 and MASP3 mRNAs. Similar results were obtained in four other glioma lines. In addition, mRNAs for C1r, C1s, C2, C3, C4, C5 and C6 were also detected in T98G cells, but very low amount of mRNAs for C1q and MBL. MBL mRNA was seen in two of the other glioma cell lines. An ELISA of culture supernatants showed that T98G cells secreted a considerable amount of MASP-1 and MASP-3 proteins. SDS–PAGE and immunoblotting analyses showed the secreted H-ficolin/Hakata antigen, MASP-1 and MASP-3 to be 34, 81 and 105 kDa in size respectively, similar to their serum counterparts. Since the glioma cells used are derived from astrocytes, this suggests that human astrocytes may be a source of some components of the LCP in the brain.

Keywords: complement, lectin, local immunity


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The complement system in blood functions as a major mediator of innate immune defense against invading microorganisms. It is activated in three ways via the classical, the alternative and the lectin complement pathways (LCP). The LCP is an antibody-independent cascade initiated by the binding of mannose-binding lectin (MBL) and its associated enzymes (MBL-associated) serine proteases (MASP) to cell-surface carbohydrate moieties (1). The molecular interactions leading to MASP activation are not clearly understood. MASP probably bind close to the N-terminal region of the collagen-like domain of MBL (2). This region of MBL forms a central core that links trimeric subunits to form bouquet-like oligomers. Three types of human MASP (MASP-1, MASP-2 and MASP-3) have been identified (36). MASP-1 was identified as a C1r/C1s-like serine protease which weakly cleaves C2 and C3 (79). Subsequently, MASP-2, with high homology to MASP-1, was isolated, and shown to activate complement via cleavage of C2 and C4 (5). These substrate specificities between MASP-1 and MASP-2 were confirmed using the recombinant proteins (9). Although the MBL–MASP-2 complex containing small MBL-associated protein sMAP works efficiently on the LCP activation, most probably MASP-1 could be also involved in the LCP with cleaving activities of C2 and C3. Recently, MASP-3 was isolated from plasma and cloned by PCR, and shown to consist of the same heavy chain/A chain as MASP-1, but a different light chain/B chain (6). Thus, MASP-1 and MASP-3 may have both shared and unique features. MASP1, MASP2 and MASP3 mRNAs are expressed mainly in liver, and the resulting proteins are secreted into the blood. MASP are not only associated with MBL, but also form complexes with members of the ficolin family proteins, L-ficolin/P35 and H-ficolin/Hakata antigen, and ficolin–MASP complexes are capable of activating the LCP (7,10,11). The ficolin polypeptide chain consists of a short N-terminal domain, a middle collagen-like domain that forms a triple helix and a C-terminal fibrinogen-like domain (12,13). These structural features are very similar to those of the collectins, such as MBL, conglutinin, collectin-43, and surfactant proteins A and D (13,14), but for the presence of a C-type carbohydrate recognition domain in the collectins. Ficolins have been identified in mammals, including human (13,1618), rodents (19), pig (12,20) and hedgehog (21) with tissue-specific distributions. They are also identified in the invertebrate, ascidian (Halocynthia roretzi) (22). Thus, in addition to MBL, L-ficolin/P35 and H-ficolin/Hakata antigen were involved in LCP activation.

There are indications that neurons are one of the most abundant sources of complement components (2325) and it is thought that neurons are responsible for local immunity by producing these proteins. We think it is possible that the LCP also plays an important role in local immunity in the brain.

To assess the importance of the LCP in local immunity, a variety of cell lines were tested for expression of complement proteins by RT-PCR. In this study, we focus particularly on glioma cell lines as a model for nerve cells and we discuss the significance of the LCP components (MASP and ficolins) in the brain.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies
Monospecific polyclonal anti-C2, -C3 and -C4 antibodies were obtained by immunization of rabbits with the respective purified human proteins. Rabbit polyclonal anti-C1r and -C1s antibodies were obtained from Calbiochem (Darmstadt, Germany), and goat polyclonal anti-C5 and -C6 antibodies from Serotec (Oxford, UK) and Cytotec (San Diego, CA) respectively. Monoclonal anti-MASP1/3 antibodies 1E2 and 2B11 were described previously (26). Hybridomas producing anti-H-ficolin/Hakata antigen antibody (4H5) and rabbit polyclonal anti-H-ficolin/Hakata antigen antibody were kindly provided by Dr N. Hamasaki (Kyushu University, Fukuoka, Japan). Rabbit polyclonal anti-MASP-3 antibody was prepared by immunizing with the peptide containing the C-terminal 20 amino acids of MASP-3 light chain. Biotin-conjugated rabbit anti-mouse Ig, biotin-conjugated goat anti-rabbit Ig and biotin-conjugated swine anti-goat Ig were purchased from Dako Japan (Kyoto, Japan).

To confirm the specificity of 1E2, 2B11 and anti-MASP-3 antibodies, an immunoblot analysis was performed (Fig. 1). The serum fraction containing MASP-1 and MASP-3 was prepared as described elsewhere (7). 1E2, 2B11 and anti-MASP-3 antibodies all reacted with serum-derived MASP-3. 1E2 and 2B11 further reacted with MASP-1, suggesting that 1E2 and 2B11 recognizes the heavy chain, which MASP-1 shares with MASP-3. The weaker staining of MASP-3 than of MASP-1 in the figure is due to the presence of more MASP-1 than MASP-3 on the blot.



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Fig. 1. Reactivities of the mAb 1E2 and 2B11 and the polyclonal anti-MASP-3 antibodies using serum-derived MASP-1 and MASP-3. MASP-1 and MASP-3 were purified from serum, and analyzed by immunoblotting with the monoclonal anti-MASP-1 antibodies, 1E2 and 2B11, and polyclonal anti-MASP-3 antibodies. Molecular weight markers (LMW and HMW) are given on the left-hand side, and the positions of MASP-1 and MASP-3 are given on the right-hand side of the figure. Both 2B11 and 1E2 are found to recognize MASP-1 and MASP-3.

 
Cell lines
The characteristics and sources of the cell lines used are given in Table 1. Cells were grown in 5% CO2 at 37°C in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml streptomycin and 100 U/ml penicillin.


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Table 1. Characteristics of the cell lines used
 
Human liver sample
Human liver was resected surgically due to the presence of a hepatoma. The sample used was obtained from a portion unaffected by the cancer. The prepared liver sample expressed the tested components except M-ficolin/P35-related and was used as a positive control.

Preparation of monocytes
Briefly, human peripheral mononuclear cells were isolated from blood on Ficoll-conray gradients (27) and monocytes were separated from lymphocytes by adherence to plastic culture dishes (28). The adherent cells were used as monocytes.

Extraction of total RNA and cDNA preparation
Total RNAs were extracted from cells (cultured with FCS) by the guanidinum isothiocyanate method (29), the concentrations were determined by absorbance at 260 nm and the RNAs were analyzed by electrophoresis on 1% agarose gels. cDNA was prepared from an equal amount of total RNA with Superscript II reverse transcription (Gibco/BRL) according to the supplier’s protocol. A amount of 0.5 µg of total RNA per each sample was used equally.

RT-PCR
PCR was carried out using AmpliTaq DNA polymerase (Gibco/BRL) according to the recommendations of the supplier. For RT-PCR, cDNA equivalent to 0.5 µg of total RNAs per each sample was used equally. The C1q (358 bp), C2 (215 bp), C3 (186 bp), C4 (256 bp), C5 (315 bp), C6 (338 bp), C7 (248 bp), C8 (258 bp), C9 (190 bp) and B (426 bp) oligonucleotide primers used for RT-PCR have been described elsewhere (30,31) (the size of the product is given in the parenthesis). PCR primers used for MASP1, MASP2, MASP3, MBL, H-ficolin/Hakata antigen, L-ficolin/P35, M-ficolin/P35-related, C1r, C1s and G3PDH were designed using PRIMER3 software in BIOSUPPLYNET and are listed in Table 2. The PCR conditions used for L-ficolin/P35 and M-ficolin/P35-related protein were as follows: 1 cycle of denaturation for 2 min at 94°C; five cycles at 94°C for 30 s, annealing at 72°C for 1 min and extension at 68°C for 2 min; five cycles at 94°C for 30 s, annealing at 70°C for 1 min and extension at 68°C for 2 min; 35 cycles at 94°C for 30 s, annealing at 68°C for 1 min and extension at 68°C for 2 min, and final elongation at 68°C for 7 min. The PCR conditions used for MASP1, MASP2, MASP3, MBL, H-ficolin/Hakata antigen, C1r and C1s were as follows: 1 cycle of denaturation step for 2 min at 94°C; five cycles at 94°C for 30 s, annealing at 65°C for 1 min and extension at 68°C for 2 min; five cycles at 94°C for 30 s, annealing at 60°C for 1 min and extension at 68°C for 2 min; 35 cycles at 94°C for 30 s, annealing at 48°C for 1 min and extension at 68°C for 2 min, and final elongation at 68°C for 7 min.


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Table 2. Sequences of primers and the expected sizes of the products
 
PCR was performed in a PCR Thermal Cycler MP (Takara, Tokyo, Japan). PCR products were analyzed by electrophoresis on 2% agarose gel. Relative optical density units (shown in the figures) represent ethidium bromide gel band intensities as determined by quantitative analysis using Image software (Scion, Frederick, MD).

Detection of MASP-1 and MASP-3 by a sandwich ELISA
Aliquots of 100 µl of culture supernatant per well were used for detecting MASP-1 and MASP-3. The supernatants used were prepared in the presence of FCS because of no interference by FCS in the ELISA. A sandwich ELISA was performed using a published procedure (26). The mAb (IE2 and 2B11) used recognize the heavy chain common to both MASP-1 and MASP-3. Thus, the ELISA indicates the sum of MASP-1 and MASP-3 proteins secreted (Fig. 1).

Preparation of H-ficolin/Hakata antigen-enriched fraction
Briefly, H-ficolin/Hakata antigen was enriched from normal serum by affinity chromatography using anti-H-ficolin/Hakata antigen (4H5)-coupled Sepharose beads. The eluted material was used as a positive control for H-ficolin/Hakata antigen.

Isolation of MASP-1 and MASP-3, and immunoblotting of MASP-1, MASP-3, H-ficolin/Hakata antigen, C1r, C1s, C2, C3, C4 and C5
Confluent T98G cells were washed extensively with RPMI 1640 and cultured for 48 h in RPMI 1640 without FCS. The supernatants prepared in the absence of FCS were used in both the isolation of MASP-1 and MASP-3, and immunoblot analyses for the detection of complements and H-ficolin/Hakata antigen (Figs 6 and 8). Prepared in the presence of FCS, even IE2-column-purified MASP-1 and MASP-3 contained FCS-derived non-specific proteins, and the representative bands were not detected in the immunoblot analyses because of the higher concentration and similar sizes of FCS-derived proteins. The supernatants were collected, centrifuged at 500 g for 10 min and filtered through a 0.22-µm filter. The filtrate was used as T98G supernatant. T98G supernatants were concentrated 1000-fold using a Ultrafree-15 centrifugal filter device (Millipore, Bedford, MA) for immunoblotting of H-ficolin/Hakata antigen, C1r, C1s, C2, C3, C4 and C5.



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Fig. 6. SDS–PAGE (A) and immunoblot (B and C) analyses of the material purified on anti-MASP-1/-3 antibody-coated beads from T98G supernatants. Samples were processed as described in Methods. The blots were immunostained with 1E2 (B) or anti-MASP-3 (C). Lane 1, serum-derived MASP-1 and MASP-3; lane 2, T98G-derived MASP-1 and MASP-3. On the left-hand side are mol. wt markers, and positions of MASP-1 and MASP-3 are given on the right-hand side.

 
T98G supernatants (prepared without FCS) were applied to beads coupled with the monoclonal anti-MASP1/3 antibody IE2. After washing with 10 mM Tris–HCl, pH 8.0 containing 150 mM NaCl and 100 µM pefablock (Pentepharm AG, Basel, Switzerland), MASP-1 and MASP-3 were eluted with 50 mM glycine–HCl, pH 2.2 containing 150 mM NaCl. The eluates were neutralized with 1 M Tris–HCl, pH 8.0 and concentrated 1000-fold using a Ultrafree-15 centrifugal filter device. Aliquots of concentrated supernatants were electrophoresed on 7.5 or 10% SDS–PAGE slab gels under reducing conditions and electrotransferred onto Immobilon-P membranes (Millipore). The blots were saturated with Blockace (Dainippon, Osaka, Japan), incubated with primary antibody and with biotin-conjugated secondary antibodies. After extensive washing, the blots were incubated with peroxidase-conjugated avidin–biotin complex (Vectastain ABC kit; Vector, Burlingame, CA). Finally the blots were developed using a Konica Immunostaining HRP-1000 Kit (Seikagaku, Tokyo, Japan).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Detection of complement components by RT-PCR
RT-PCR was used to examine the constitutive expression of complement component mRNAs in human liver and peripheral monocytes. Primers designed for complement component (C1r, C1s, C1q, C2, C3, C4, C5, C6, C7, C8, C9, B, MASP1, MASP2, MASP3, MBL, L-ficolin/P35, M-ficolin/P35-related and H-ficolin/Hakata antigen) mRNAs were used. G3PDH was included as an internal standard to ensure that the levels of cDNAs in the different samples were comparable. Figure 2 shows that the mRNAs of all the complement components, with the exception of M-ficolin/P35-related, could be detected in human liver. M-ficolin/P35-related could be detected in peripheral monocytes (used as a positive control of M-ficolin/P35-related). The sizes of all were consistent with those predicted (see Methods). The faint band in the C1q lane in T98G cells is an artifact. The levels of internal standard G3PDH mRNAs in the 21 tested lines and controls were comparable (Fig. 3A), indicating that the levels of mRNAs detected reflect the cell-specific expression levels in the experiments.



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Fig. 2. Polaroid photographs of typical ethidium bromide-stained gels demonstrating PCR products for complement component mRNAs in human liver and T98G cells. The marker lane contains the following size markers (from the top): 1353, 1078, 872, 603, 310, 281,271, 234 and 194 bp ({phi}X174 HaeIII digest). P35, L-ficolin/P35; P35r, M-ficolin/P35-related; HA, H-ficolin/Hakata antigen. Only for M-ficolin/P35-related, human peripheral monocytes instead of human liver were used as a positive control.

 


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Fig. 3. Bar graph showing the relative levels of G3PDH (A), MBL, L-ficolin/P35, M-ficolin/P35-related and H-ficolin/Hakata antigen (B), MASP1, MASP2 and MASP3 (C), and C1r and C1s (D) mRNAs in extracts of 17 cell lines (T lymphocytes: Molt 3, Molt 4 and MT-2; B lymphocytes: Raji, Ramos, Ramos/B and Daudi; monocytic: HL60, THP1 and U937; basophilic: KU812F; NK-like: YT; glioma/astrocytic: T98G; lung-derived: TKB1; hepatocyte: HepG2; cervix-derived: HeLa; colon-derived: HT29) and in human liver or in monocytes (only for M-ficolin/P35-related) (Liver/Mono). An amount of 0.5 µg of total RNAs per each sample was used equally. Values of four to six experiments are averages with bars indicating SD. Abbreviations are as in the legend to Fig. 2.

 
Expression of MBL, L-ficolin/P35, M-ficolin/P35-related and H-ficolin/Hakata antigen mRNAs
The expression of mRNAs for recognition molecules in the LCP was examined. To examine which type of cells express MBL, L-ficolin/P35, M-ficolin/P35-related and H-ficolin/Hakata antigen mRNAs, RT-PCR was performed on 17 cell lines (Fig. 3B). Expression of L-ficolin/P35 and H-ficolin/Hakata antigen mRNAs was detected in lymphoid, myeloid as well as non-myeloid cells. In contrast, MBL mRNA expression was strongly observed in HepG2 and MT-2. M-ficolin/P35-related mRNA expression was negligibly low in all the cell lines tested. In T98G cells, a relatively high level of H-ficolin/Hakata antigen mRNA was detected, whereas the other lectin mRNAs were at low levels. MBL, L-ficolin/P35 and H-ficolin/Hakata antigen mRNAs are detected in human liver, but not M-ficolin/P35-related, while M-ficolin/P35-related mRNA was detectable in normal peripheral monocytes.

Expression of MASP1, MASP2, MASP3, C1r and C1s mRNAs
To study which type of cells express MASP1, MASP2, MASP3, C1r and C1s mRNAs, RT-PCR was performed with 17 cell lines (Fig. 3C and D). MASP mRNAs were expressed broadly and at varying levels by 11 of the 17 cell lines (Fig. 3C). There was a tendency for non-lymphoid, non-myeloid lines such as T98G, TKB1, HepG2 and HeLa to express MASP1 and MASP3 mRNAs at relatively high levels, while lymphoid and myeloid lines (especially monocytic lines) expressed only negligible levels of MASP mRNAs. It was of interest that a glioma line, T98G, expressed MASP1 and MASP3 mRNAs at levels as high as liver, which is the main source of serum complement components. Of the cell lines tested, only HepG2 expressed MASP2 mRNA at a significant level. It seems likely that hepatocytes are specific producers of MASP2. HeLa and TKB-1 also expressed relatively large amounts of MASP3 mRNA. The C1q-associated serine proteases, C1r and C1s, like MASP mRNAs, were broadly expressed (Fig. 3D). Monocytic cell lines expressed lower levels of C1r and C1s mRNAs.

Expression of components of the LCP in glioma cell lines
The finding that T98G cells express high levels of MASP1 and MASP3 mRNAs suggests the possibility that the expression of the LCP components may be common characteristics for astrocytes. To assess this, four other glioma cell lines derived from astrocytes, A7, A172, 1321N1 and U251, were examined for expression of the mRNAs for components of the LCP. The results shown in Fig. 4(A) show that comparable levels of G3PDH mRNAs were recovered from the glioma lines. On the other hand, the glioma cell lines were found to express MASP1 and MASP3 mRNAs, but negligible amounts of MASP2 mRNA (Fig. 4C). However, the levels of expression in A7, A172, 1321N1 and U251 cells were lower than in T98G cells. There was no similarity in the expression pattern of mRNAs for MBL, L-ficolin/P35, M-ficolin/P35-related and H-ficolin/Hakata antigen, among the glioma lines (Fig. 4B), with the exception that the expression of M-ficolin/P35-related mRNAs was negligible in all of them. Of five glioma lines tested, A172 and 1321N1 expressed MBL mRNA at a relatively high level. The results suggest that astrocytes express the components of the LCP.



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Fig. 4. Bar graphs showing relative levels of the following mRNAs in extracts of five glioma cell lines (T98G, A7, A172, U251 and 1321N1) and human liver or in monocytes (only for M-ficolin/P35-related) (Liver/Mono): G3PDH (A), MBL, L-ficolin/P35, M-ficolin/P35-related and H-ficolin/Hakata antigen (B), and MASP1, MASP2 and MASP3 (C). An amount of 0.5 µg of total RNAs per each sample was used equally. Values of four to six experiments are averages with bars indicating SD. Abbreviations are the same as in Fig. 2.

 
Secretion of MASP-1 and MASP-3 proteins by glioma cell line T98G
To examine whether MASP-1 and MASP-3 proteins are secreted by T98G (MASP1+/MASP2/MASP3+), HeLa cells (MASP1/MASP2/MASP3+) and TKB-1 (MASP1/MASP2/MASP3+) consistent with their levels of mRNA expression, a sandwich ELISA was performed using the supernatants prepared with FCS (Fig. 5). Since the mAb (1E2 and 2B11) against MASP-1 which we used recognize the heavy chain which is common to both MASP-1 and MASP-3, both of them are detected with this ELISA. As shown in Fig. 5, T98G cells secreted significant levels of MASP-1 and MASP-3, while HeLa and TKB-1 cells secreted only very low levels, suggesting that, in HeLa and TKB-1 cells, MASP-3 protein might be secreted only in response to certain stimuli.



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Fig. 5. Bar graph showing the levels of secreted MASP-1/MASP-3. Supernatants of T98G (T98G sup), HeLa (HeLa sup) and TKB-1 (TKB1 sup) cultures were collected and analyzed for MASP-1/-3 by ELISA. RPMI 1640 containing 10% heat-inactivated FCS (Medium) and serum-derived MASP-1 and MASP-3-enriched fraction (pMASP-1/-3) were used as negative control and positive controls respectively. Values of three experiments are averages with bars indicating SD.

 
The level of MASP-1/MASP-3 in serum was estimated to be ~6 µg/ml (26) and it was ~3 µg/ml (~100 ng/106 cells/day) in the T98G supernatants prepared with FCS, suggesting similarly functional levels of production. Since T98G cells produce MASP-1/MASP-3 proteins, they could be good models for studying its production in the brain.

Isolation of MASP-1 and MASP-3 proteins from T98G culture supernatant
Since 1E2 recognizes both MASP-1 and MASP-3 (Fig. 1), the above ELISA shows the combined production of MASP-1 and MASP-3. To confirm that both MASP-1 and MASP-3 are produced by T98G and to define the size of the MASP-3, MASP-1 and MASP-3 proteins were isolated from T98G supernatants by IE2-coupled Sepharose beads. Since FCS involvement in the supernatants gave non-specifically bound proteins in the affinity chromatography, the supernatants were prepared without FCS (see Methods). The eluted material was analyzed by SDS–PAGE under non-reducing conditions (Fig. 6A) and immunoblotting (Fig. 6B and C), which showed MASP-1 and MASP-3 to be 81 and 107 kDa respectively. A 107-kDa band was also immunostained by the anti-MASP-3 light chain C-terminal peptide antibody (Fig. 6C). The obtained MASP-1 and MASP-3 were identical sizes to serum counterparts and would be functionally active. More MASP-3 than MASP-1 was secreted by the T98G cells in accordance with the relatively higher MASP3 mRNA expression.

Productive expression of C1r, C1s, C2, C3, C4, C5, C6 and H-ficolin/Hakata antigen in T98G cells
Tested for expression of the lectin mRNAs, T98G cells expressed a clear level of mRNA for H-ficolin/Hakata antigen and lower levels of other lectins as shown in Fig. 3. Furthermore, we analyzed whether the T98G cells produce other relevant complement factors as control proteins. Using RT-PCR, C1r, C1s, C2, C3, C4, C5 and C6 mRNAs were detected at significant levels in T98G cells (Fig. 7), but no mRNA for C1q could be detected.



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Fig. 7. Bar graph showing the relative levels of C1r, C1s, C1q, C2, C3, C4, C5, C6, C7, C8, C9 and B mRNAs in extracts of human liver and T98G cells. An amount of 0.5 µg of total RNAs per each sample was used equally. Values of four experiments are averages with bars indicating SD.

 
Thus, it would be expected that T98G cells produce these proteins as well as H-ficolin/Hakata antigen in accordance with the expression of mRNAs. This was confirmed by immunoblot analyses which revealed the secretion of C1r, C1s, C2, C3, C4, C5, C6 and H-ficolin/Hakata antigen by T98G cells (Fig. 8). The supernatants were prepared without FCS since comparably higher amounts and similar sizes of serum proteins interfere with the detection, and highly concentrated because of the lowered secretion by no FCS culture. It seems that at least the early complement components (except C1q) are produced at effective levels by T98G cells.



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Fig. 8. Immunoblot analysis of complement components secreted by T98G cells. The supernatants prepared in the absence of FCS were concentrated 1000-fold and processed as described in Methods. Lane 1, normal serum (x20 diluted); lane 2, concentrated T98G supernatant (1000-fold). The bands for C3, C4 and C5 are {alpha}, {gamma} and {alpha} chains respectively.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immunity is divided in innate and acquired immunities. The innate immune system seems to be older than the acquired immune system. The innate immune system is the older, more primitive recognition system with less specificity. The complement system functions as the innate immune system to clear deleterious invaders. The blood–brain barrier is a major protective mechanism for the central nervous system against circulating bacteria. The brain cells can synthesize full lytic complement components and complement receptors (32), suggesting the importance of the complement system within the blood–brain barrier. It is known that the classical and the alternative complement pathways contribute to local immunity, which is probably the first line of immune defense. However, little is known about the contribution of the LCP to local immunity. To address this, using RT-PCR we examined the expression of mRNAs for components of the LCP in a variety of cell lines. This analysis showed that the glioma cell line T98G expresses MASP1 and MASP3 mRNAs; ELISA and immunoblotting confirmed that both proteins are synthesized and secreted by T98G cells. The estimated sizes of MASP-1 and MASP-3 were 81 and 107 kDa respectively. As MASP-3 in the T98G cells was found to be produced in larger amounts than MASP-1, it could be speculated that MASP-3 might have more important roles than MASP-1 in the brain. Productive expression of H-ficolin/Hakata antigen by T98G cells was also observed, although, unexpectedly, very low MBL and MASP2 mRNA levels were detected in T98G cells. The early complement components were detected in T98G supernatants, but mRNAs for the late components were negligibly low. However, since human cerebrospinal fluid contains enough C1 to C9 to give hemolytic activity (33), and MASP-1 cleaves C2 and C3 (79), the LCP might still work within the blood–brain barrier. H-ficolin/Hakata antigen was not detected in material from T98G cells purified on anti-MASP-1/-3 antibodies, suggesting that H-ficolin/Hakata antigen does not associate with MASP-1 and MASP-3 (not shown). There is still a chance that H-ficolin/Hakata antigen can form a complex with MASP after H-ficolin/Hakata antigen is bound to the surface of xenobiotics.

In this study, we focused on gliomas/astrocytes, but it is also very interesting that MASP3 mRNA was expressed at comparably high levels in HeLa and TKB-1 cells. MASP3 expression in these cell lines could contribute to local immunity in lung and cervix because they are located at the front line of the immune defense mechanism. We detected high levels of MASP2 mRNA only in HepG2, whereas the others tested express very low MASP2 mRNA levels. Thus, it is likely that MASP2 expression is restricted to hepatocytes. The differences in expression of MASP1, MASP2 and MASP3 probably reflect the functional differences of the derived proteins.

The present study shows that cells do not always express both lectin and MASP, although a complex of these is expected to be the initiating factor of the LCP. Although it is the MBL–MASP-2 complex that activates the LCP efficiently via C2 and C4 cleavages, T98G cells express very low MBL and MASP2 mRNAs levels, unexpectedly. Instead, the other combination, H-ficolin/Hakata antigen–MASP-1/MASP-3, was obtained in T98G cells. The difference of the substrate specificity between MASP-1 and MASP-2 is an activity in which C3 and C4 are mainly cleaved. It is likely that the molecular-evolutional appearance periods of MASP correlate with the periods of the substrate molecules (C3 and C4) (34). From this point of view, C3 and MASP-1/MASP-3 molecules seem to be older than C4 and MASP-2 molecules, suggesting the LCP initiation consists of two steps dependent on the molecular evolution (C3/MASP-1 and C4/MASP-2). At present, MASP-3 function is unclear, but the high expression of MASP-3 in astrocytes indicates that MASP-3 is involved in other functions that could be important in the brain.

On the other hand, lectin mRNAs in the cell lines showed different expression spectra. L-ficolin/P35 and H-ficolin/Hakata antigen are expressed broadly, whereas MBL mRNA is expressed in MT-2 and HepG2, and M-ficolin/P35-related mRNA is negligibly low in all cell lines tested. Thus, the expression of MBL and M-ficolin/P35-related is more restricted than H-ficolin/Hakata antigen and L-ficolin/P35. Expression of L-ficolin/P35 and H-ficolin/Hakata antigen in T, B and monocytic cell lines is expected to contribute to cellular immunity. Even in gliomas tested, variable expression of the lectins was obtained, suggesting that the fine differences (such as differentiation and activation) among the gliomas influence the lectin expression. Lectins in themselves would be molecules independent of complement and might not always be regulated dependently on it. It would be acceptable that lectins as a recognition molecule are variably expressed since a variable lectin–MASP combination could possibly produce LCP activation (10,11). The important feature of a model cell line for studying the LCP would be to express at least both one of the three lectins (MBL, L-ficolin/P35 and H-ficolin/Hakata antigen) and either of MASP-1 and MASP-2.

In conclusion, human glioma T98G cells express H-ficolin/Hakata antigen, MASP-1 and MASP-3. This suggests that the LCP functions as a local innate immune system in the brain. It is expected that the role of the LCP components produced locally by HeLa and TKB1 as well as T98G is to work independently of serum components right at the invasion since cervix/HeLa and lung/TKB-1 locate at the front of the defense. MASP-1 seems to be the more primitive molecule to initiate the LCP than MASP-2. Within the blood–brain barrier, such primitive MASP-1(-3) would mainly work as local immunity. T98G cells should be convenient models for studying the production of MASP-1 and MASP-3 by human astrocytes in the central nervous system. It is anticipated that further studies will clarify the functions of MASP-3 in brain as well as in plasma.


    Acknowledgements
 
We thank Dr J. C. Jensenius for critical reading and Dr H. M. Schulman for English editing.


    Abbreviations
 
LCP—lectin complement pathway

MBL—mannose-binding lectin

MASP—MBL-associated serine protease

In this paper, MASP proteins are designated as MASP-1, MASP-2 and MASP-3, whereas MASP genes are designated as MASP1, MASP2 and MASP3.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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