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. 2001 Oct;104(2):207–214. doi: 10.1046/j.0019-2805.2001.01280.x

Characterization of glycosylphosphatidylinositol-anchored decay accelerating factor (GPI-DAF) and transmembrane DAF gene expression in wild-type and GPI-DAF gene knockout mice using polyclonal and monoclonal antibodies with dual or single specificity

Takashi Miwa *, Xiujun Sun *, Rieko Ohta †, Noriko Okada †, Claire L Harris ‡, B Paul Morgan ‡, Wen-Chao Song *
PMCID: PMC1783299  PMID: 11683961

Abstract

Decay-accelerating factor (DAF, CD55) is a glycosylphosphatidylinositol (GPI)-linked membrane inhibitor of complement activation. While human and other mammalian species contain only one DAF gene, two distinct DAF genes, referred to as GPI-DAF and transmembrane (TM)-DAF, respectively, have been identified in the mouse. Using several independently generated monoclonal and polyclonal antibodies, either with dual or single specificity for GPI-DAF and TM-DAF gene products, we have examined the expression of the two DAF genes in tissues of the wild-type and a strain of knockout mouse whose GPI-DAF gene has been inactivated. By fluorescence-activated cell sorting (FACS) analysis, we found that DAF protein is present on the wild-type mouse erythrocytes and lymphocytes but no signal was detectable on the same cells of GPI-DAF gene knockout mice. Both T and B lymphocytes and splenic macrophages express the GPI-DAF gene but the expression level is higher on B lymphocytes than on T lymphocytes. Within the T cell population, both CD4+ and CD8+ T cells are positive. DAF protein was detected by immunohistochemistry at high levels on wild-type mouse spermatids and mature sperm. In contrast, only mature sperm stained positive in the GPI-DAF gene knockout mouse testis, suggesting that GPI-DAF but not the TM-DAF gene is expressed on spermatids. Examination of the fetoplacental unit at the day 7·5 stage revealed that GPI-DAF but not the TM-DAF gene is expressed in the maternal decidua cells surrounding the trophoectoderm of the embryo. No DAF expression was detected on trophoblast or the embryo proper. These findings suggest that although the TM-DAF gene is irrelevant on mouse blood cells, the two DAF genes may have different roles in germ cell development and/or mature sperm function. Because complement receptor 1-related gene/protein y (Crry) has been shown to be expressed on early mouse embryos, the complete lack of GPI-DAF and TM-DAF gene expression in early mouse development may explain the observed sensitivity of Crry-deficient embryos to maternal complement attack.

Introduction

The complement system is composed of a series of plasma proteins which play an important role in the innate immune response to invading pathogens.1 Activated complement releases pro-inflammatory anaphylatoxins and generates a membrane attack complex that causes the direct lysis of micro-organisms it encounters.1 To avoid inadvertent complement-mediated autologous tissue damage, host cells, particularly those that have close contact with plasma such as erythrocytes and endothelial cells, express a number of membrane-bound inhibitors of complement activation.1–3 One of the best-characterized membrane complement inhibitors in humans is decay-accelerating factor (DAF, CD55).3,4 DAF attaches to the cell surface via a glycosylphosphatidylinositol (GPI) anchor and functions by preventing the formation and accelerating the decay of the biomolecular C3 convertase complexes of both the classical and alternative pathways of complement.4 By inhibiting the pivotal C3 cleavage step in the complement activation cascade, DAF is thus regarded as a central molecule in preventing autologous complement attack.

The important role that DAF plays in protecting self tissues from complement damage is illustrated by the human haematological disorder, paroxysmal nocturnal haemoglobinuria (PNH), a syndrome characterized by tendency of autologous complement-mediated lysis of erythrocytes. PNH is caused by deficiency of DAF and a second complement inhibitor, CD59, on affected blood cells.5–7 In addition to being a well-established complement regulator, there is evidence to suggest that DAF, as a GPI-anchored cell surface molecule, may also function as a signalling molecule.8 For example, it has been demonstrated that cross-linking of DAF by antibodies led to lymphocyte activation.9 Moreover, DAF has recently been identified as a ligand for a seven transmembrane, receptor-like leucocyte antigen CD97 that becomes rapidly expressed on lymphocytes after cell activation.10–12

To facilitate in vivo analysis of DAF function in animal models, DAF homologues from various mammalian species have been characterized.3 Interestingly, while only one DAF gene is known to exist in the human and every other animal species studied, the mouse has been shown to contain two separate DAF genes in its genome.13–15 These two genes are highly homologous in their genomic structure and sequence and are arranged in tandem on mouse chromosome 1.14 One of the two genes was predicted to encode a GPI-anchored DAF molecule similar to the human DAF while the other gene was predicted to encode a molecule that inserts into the membrane via a transmembrane (TM) domain.14 These two DAF genes have been referred to as the GPI-DAF and TM-DAF gene, respectively. However, subsequent studies have suggested that mRNA encoding a putative GPI-anchored form of DAF could also be transcribed from the TM-DAF gene.16 Northern blotting and reverse transcription–polymerase chain reaction (RT–PCR) evidence obtained previously has suggested that the GPI-DAF gene is expressed broadly in various mouse tissues, including the testis, whereas the expression of the TM-DAF gene appears to be restricted to the testis with possible low-level expression in the spleen.13,14,16 On the other hand, little is known about the relative expression of GPI-DAF and TM-DAF gene on mature mouse erythrocytes and it remains unclear whether the two genes have overlapping or discrete expression patterns within the mouse testis. Pertinent to the former question, it has been observed that DAF on mouse erythrocytes was resistant to phosphatidylinositol-specific phospholipase C (PIPLC) cleavage17 and that a GPI-DAF gene knockout mouse is phenotypically normal with regard to its erythrocytes being adequately protected from autologous complement attack.18 Surprisingly, deletion of the rodent-specific membrane C3 convertase inhibitor complement receptor 1-related gene/protein y (Crry) proved to be lethal as a result of maternal complement attack on early embryos.19 In order to understand the relative expression and roles of GPI-DAF and TM-DAF genes in comparison with that of Crry on mouse blood cells and reproductive tissues, we have examined the expression of GPI-DAF and TM-DAF genes in several tissues of interest in wild-type and the GPI-DAF gene knockout mice using a panel of monoclonal antibodies (mAb) and polyclonal antibodies, with either single or dual specificity for the two gene products.

Materials and methods

Cell culture and DAF cDNA transfection experiments

Human embryonic kidney 293 (HEK 293) cells were cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS).11 Expression plasmids for mouse GPI-DAF cDNA used in HEK 293 cell transfection were constructed in the eukaryotic expression vector pCDNA3 (Invitrogen, Carlsbad, CA). GPI-DAF cDNA (nucleotide 1 to 133415) was subcloned into pCDNA3 at EcoRI sites. For transfection experiments, HEK cells were seeded at 60% confluency in 100 mm dishes and transfected with 5 µg DAF cDNA plasmids or empty pCDNA3 vector using Lipofectamine (Gibco/Life Technologies, Grand Island, NY) by following the manufacturer's instruction. Two days after transfection, G-418 (Gibco/BRL) was added to the cell culture medium at 300 µg/ml to select transfected cells. The selection process was allowed to continue for 17 days. Expression of GPI-DAF on G-418 resistant cells was examined by fluorescence-activated cell sorting (FACS) analysis using a hamster anti-mouse DAF mAb Riko-3.20 A high-expressing cell population in the GPI-DAF HEK transfected cells was sorted by FACS. Generation of stable Chinese hamster ovary (CHO) cell lines expressing the mouse GPI-DAF cDNA or the TM-DAF cDNA has been described previously.20

FACS analysis

FACS analysis was performed on DAF-transfected HEK cells, DAF-transfected CHO cells and mouse erythrocytes and splenocytes. Sources of the antibodies were as follows: a rabbit polyclonal anti-mouse DAF was raised using purified mouse erythrocyte DAF as an antigen as previously described.17 Details for the generation of two hamster anti-mouse DAF mAb, Riko-3 and Riko-4, were described previously.20 Riko-3 recognizes both GPI-DAF and TM-DAF whereas Riko-4 is specific for GPI-DAF. A third mAb anti-mouse DAF, 2C6.5, was generated in the rat using GPI-DAF-transfected CHO cells as antigens. Screening of DAF-reactive antibodies was carried out by comparative enzyme-linked immunosorbent assay (ELISA) assays using non-transfected and transfected CHO cells according to a previously established protocol.21 For analysis of HEK cells and CHO cells, cells stably transfected with GPI-DAF or TM-DAF and growing in the log phase were harvested by trypsinization, washed in phosphate-buffered saline (PBS) and re-suspended in PBS at a final concentration of 2 × 107/ml. For analysis of erythrocytes, blood samples (30–40 µl) were collected by tail vein bleeding from wild-type and GPI-DAF knockout mice (B6/129 background) into 1 ml Ca2+,Mg2+-free PBS. Total blood cells were collected by centrifugation at 1000 g and washed several times in PBS. The cells were finally re-suspended in PBS at 5 × 107/ml. Preparation of erythrocyte-free splenocytes was carried out as described.11 Briefly, freshly isolated spleen was macerated and ground in cold Hank's solution until no tissue lumps remained. The tissue mixture was then passed through a cell strainer (80 µm) and splenocytes were subsequently collected by low-speed centrifugation (500 g). Contaminating erythrocytes were removed by lysis in a buffer composed of 150 mm NH4Cl, 1 mm KHCO3 and 0·1 mm ethylenediaminetetraacetic acid (EDTA), pH 7·3. The cells were finally re-suspended in PBS at a final concentration of 1×107/ml.

For HEK, CHO and erythrocyte staining (single color-staining), cells (50 µl) were first incubated with the primary antibody at 4 C for 30 min (1 : 125 for the rabbit polyclonal antibody, 1 : 50 for Riko-3 and 1 : 10 for Riko-4 immunoglobulin G (IgG) preparations at 1 mg/ml, and 1 : 10 for a 2C6.5 IgG preparation at OD280 = 1·74). After washing several times with PBS, cells were stained with a secondary antibody for 30 min (phycoerythrin (PE)-conjugated goat anti-rabbit IgG (Sigma, St Louis, MO) diluted at 1 : 50 for the polyclonal antibody, PE-conjugated goat anti-hamster IgG (Pharmingen, La Jolla, CA) diluted at 1 : 50 for Riko-3 and Riko-4, and fluoroscein isothiocyanate (FITC)-conjugated goat anti-rat IgG (Jackson Immuno Research, West Grove, PA) diluted at 1 : 50 for 2C6.5). For splenocyte staining (double-colour staining), cells (50 µl) were first stained with Riko-3 or Riko-4 in the presence of Fc block (Pharmingen, used at 1 : 100 dilution), washed and then stained with a PE-conjugated goat anti-hamster IgG together with a FITC-conjugated anti-mouse CD3 or B220 or CD11b antibody (hamster anti-mouse CD3, rat anti-mouse B220, rat anti-mouse CD11b, all from Pharmingen). All analyses were carried out using a FACScan machine (Becton Dickinson, San Jose, CA) by gating the appropriate cell populations.

Immunohistochemistry

To prepare for immunohistochemical staining, testes from wild-type and GPI-DAF knockout mice were fixed in cold (4°) Bouin's solution overnight, dehydrated and paraffin-embedded. Paraffin sections were made at 5 µm thickness. Tissue sections were first deparaffinized in xylene and graded ethanol, washed in distilled water and then processed for immunostaining. Spleen and day-7·5 fetoplacental unit (plug day as day-0·5) were snap-frozen in OCT medium (TissueTek; Miles, Inc., Elkhart, IN) and cut in 4 µm frozen sections. The sections were fixed in acetone for 15 min. Deparaffinized testis sections or acetone-fixed spleen and placenta sections were first treated with 3% H2O2 in methanol for 15 min to quench endogenous peroxidase activity. After rinsing twice with PBS, diluted normal goat serum (1·5%) was added to the slides to reduce non-specific binding. The slides were incubated for 30 min, rinsed with PBS and the polyclonal rabbit anti-mouse DAF serum or a control non-immunized rabbit serum (both at 1 : 2000 dilution) was then added. Formation of antigen–antibody complexes was allowed to take place for 60 min at room temperature. Detection of DAF protein was achieved by using a secondary antibody coupled to a peroxidase–biotin–streptavidin detection system (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA) and with diaminobenzidine tetrahydrochloride (DAB) as the peroxidase substrate for color detection.

Results

To confirm the reactivity and specificity of the antibodies, we used several stable cell lines which express either the mouse GPI-DAF or TM-DAF cDNA. These cell lines included a HEK 293 cell line expressing the GPI-DAF, and two CHO cell lines expressing the GPI-DAF and TM-DAF, respectively. Figure 1 shows the results of FACS analysis of these cells using four different anti-mouse DAF antibodies. All four antibodies gave background staining in control (vector-transfected) HEK or CHO cells (Fig. 1a–f, shaded areas). All antibodies reacted with GPI-DAF expressed on HEK cells (Fig. 1a–d). In a previous study, it has also been determined that the polyclonal antibody and the mAb Riko-3 but not mAb Riko-4 also recognized TM-DAF expressed on CHO cells.20 As shown in Fig. 1(e,f), the mAb 2C6.5 recognized TM-DAF as well as GPI-DAF expressed on CHO cells. Thus, we have confirmed that the polyclonal antibody and the mAb Riko-3 and 2C6.5 are suitable reagents for studying the relative expression of GPI-DAF and TM-DAF gene products in mouse tissues.

Figure 1.

Figure 1

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Specificities of anti-mouse DAF antibodies. GPI-DAF cDNA-transfected HEK 293 cells (a–d) were stained with Riko-3 (a), Riko-4 (b), polyclonal anti-DAF (c), or 2C6·5 (d). GPI-DAF cDNA (e)- or TM-DAF cDNA (f)-transfected CHO cells were also stained with 2C6.5. Vector cDNA-transfected HEK 293 or CHO cells were stained as controls for background staining (shaded areas in all panels).

We then used the mAb Riko-3, Riko-4 and 2C6.5 to detect the presence of DAF protein(s) on the surface of wild-type and GPI-DAF gene knockout mouse erythrocytes. Figure 2(c,d) shows that Riko-4 (GPI-DAF-specific) detected GPI-DAF gene expression on the erythrocytes of a wild-type mouse but not of a GPI-DAF gene knockout mouse. This result is consistent with our previous Northern blot analysis of the GPI-DAF gene knockout mouse that demonstrated complete inactivation of the GPI-DAF gene.18 Notably, when mAb Riko-3 and 2C6.5 (which recognize both GPI-DAF and TM-DAF gene products) were used, a similar result was observed, i.e. clear signals were detected on wild-type erythrocytes but not on GPI-DAF gene knockout erythrocytes (Fig. 2a,b,e,f). From these observations, it can be concluded that the TM-DAF gene is not expressed on mouse erythrocytes, either constitutively or in a compensatory fashion when the GPI-DAF gene is inactivated.

Figure 2.

Figure 2

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FACS analysis of DAF expression on wild-type (+/+) and GPI-DAF knockout (−/−) mouse erythrocytes. All three monoclonal antibodies detected DAF expression on wild-type mouse erythrocytes (a, c, e) but not on GPI-DAF gene knockout mouse erythrocytes (b, d, f). Open area under the curve represents specific staining with first and secondary antibodies. Shaded area represents background staining with secondary antibody only. No specific staining was detected on the knockout erythrocytes.

Because TM-DAF mRNA message has previously been detected in the mouse spleen by RT-PCR16 and Northern blot analysis,14 we wondered whether the spleen might be a tissue where DAF proteins from both genes are made, and if that is the case whether these DAF proteins might be expressed in distinct spleen cell populations. Total splenocytes from wild-type and GPI-DAF gene knockout mice were analysed by two-coloured FACS using Riko-3 or Riko-4 for DAF expression and a T-lymphocyte or B-lymphocyte or macrophage-specific antibody for gating the appropriate cell populations. Figure 3 shows that the majority of B-lymphocytes (B220 positive) and T lymphocytes (CD3 positive) from wild-type mice stained positive for DAF. Nevertheless, a substantial percentage of CD3-positive cells appeared to be DAF-negative (Fig. 3e,g). In a subsequent experiment, CD4+ and CD8+ T lymphocytes were gated and analysed by two-coloured FACS. No difference was found in DAF staining between these two populations of T lymphocytes (data not shown). It is apparent from staining with either Riko-4 or Riko-3 that the average level of DAF protein on B cells is significantly higher than that on T cells (Fig. 2a,c,e,g). As with erythrocytes, no DAF staining was detectable on B or T lymphocytes from GPI-DAF gene knockout mice, suggesting that the TM-DAF gene is not expressed on these cells. Macrophages constitute another major cell population within the spleen and we investigated if the TM-DAF gene might be selectively expressed on spleen macrophages. As shown in Fig. 4, the majority of CD11b-positive spleen cells (gated for CD-11b staining in Fig. 4) express DAF on their surface. Once again, no DAF protein was detected on CD11b-positive cells form GPI-DAF gene knockout mice, suggesting that TM-DAF is not expressed on these cells either.

Figure 3.

Figure 3

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Two-coloured FACS analysis of DAF expression on spleen lymphocytes in wild-type (+/+; a, c, e, g) and GPI-DAF knockout (−/−; b, d, f, h) mice. B lymphocytes (a–d) and T lymphocytes (e–h) were identified using B220 and CD3 as a marker, respectively. An overwhelming majority of B220-positive cells and a majority of CD3-positive cells stained positive for DAF with either Riko-3 or Riko-4. No signal was present on GPI-DAF knockout cells.

Figure 4.

Figure 4

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Two-coloured FACS analysis of DAF expression on spleen macrophages in wild-type (+/+; a, c, e) and GPI-DAF gene knockout (−/−; b, d, f) mice. CD11b was used as a marker for gating the macrophage population. (a, b) Cells stained with a PE-conjugated secondary antibody only (goat anti-hamster IgG). DAF is expressed on the majority of CD11b-positive spleen macrophages in wild-type mice whereas no specific staining was detectable on the same cells from GPI-DAF knockout mice. Thus, although Riko-3 produced a stronger signal than Riko-4 on macrophages, this was unlikely an indication of TM-DAF gene expression on these cells. It is possible that Riko-3 may have a relatively higher affinity for the DAF protein expressed on macrophages than on lymphocytes (e.g. degrees of glycosylation of DAF may vary from cell to cell).

To determine the relative expression of the GPI-DAF and TM-DAF genes in the testis, immunohistochemical studies were carried out using a rabbit polyclonal anti-mouse DAF serum.17 Fig. 5(b,e) show that specific DAF staining was detected in the testes of both wild-type and GPI-DAF gene knockout mice. However, it is clear that the staining patterns are rather different in the two types of animals. In the wild-type mouse testis, prominent staining was detected on mature sperm in the lumen of the seminiferous tubules as well as on the inner layers of developing germ cells of the tubule which corresponded predominantly to spermatids (Fig. 5b). In contrast to this pattern, only mature sperm located in the lumen of the seminiferous tubules stained positive in the GPI-DAF gene knockout mouse testis (Fig. 5e). This suggested that GPI-DAF but not the TM-DAF gene is expressed on spermatids and that TM-DAF gene expression is restricted to mature sperm. To confirm the FACS data on the lack of TM-DAF gene expression on spleen lymphocytes and macrophages, we also performed immunohistochemical staining of spleen sections. Figure 5(c,f) show that specific and prominent staining was detected in both the red pulp and white pulp areas of the wild-type spleen but no staining was observed in the GPI-DAF gene knockout spleen.

Figure 5.

Figure 5

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Immunohistochemical staining of DAF expression in wild-type (a, b, c) and GPI-DAF knockout (d, e, f) mouse testis (a, b, d, e) and spleen (c, f). To detect DAF protein, a rabbit polyclonal antibody was used (b, c, e, f). No signal was detected in the testis (a, d) or the spleen (data not shown) when a non-immune rabbit serum was used. Positive signal was detected on late-stage developing germ cells and mature sperm in the wild-type testis (b) and on mature sperm but not developing germ cells in the GPI-DAF knockout testis (e). Both red pulp and white pulp areas stained positive in the wild-type mouse spleen but no signal was detected in the GPI-DAF knockout mouse spleen.

Human DAF has been detected in a number of studies on fetal trophoblast and is assumed to play a role in preventing fetal rejection mediated by maternal humoral or cell-mediated immunological mechanisms.22–24 To determine if one or both of the DAF genes are expressed in the fetoplacental tissues of the mouse, we isolated the fetoplacental units from wild-type and GPI-DAF knockout mice at the 7·5-day stage and performed immunohistochemistry on frozen sections of this tissue. Figure 6 shows that, within the whole fetoplacental unit, GPI-DAF gene is expressed only on a subset of cells in the maternal decidua surrounding the trophoectoderm of the embryo but not on cells of embryonic origin (Fig. 6b). No staining on corresponding cells of fetoplacental units from GPI-DAF gene knockout mice was detected (Fig. 6c), suggesting that the TM-DAF gene is neither expressed in the maternal decidua nor on the embryos of this developmental stage.

Figure 6.

Figure 6

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Immunohistochemical staining of DAF expression in day-7·5 mouse fetoplacental units (10× magnification). Positive staining was observed in the maternal decidua cells surrounding the developing embryo in the wild-type mice (b, highlighted by arrow). No staining was detected on corresponding cells in the fetoplacental unit of a GPI-DAF knockout mouse (c) or in the wild-type fetoplacental unit when a non-immune rabbit serum was used (a). EP, embryo proper.

Discussion

The role of DAF as a central complement regulator has been well established by in vitro studies but the in vivo biology of the protein remains to be fully characterized. In many ways, the mouse represents an excellent animal model for studying the in vivo functions of DAF both in normal physiology and in disease pathogenesis where complement activation is involved. However, the analysis of the in vivo function of DAF using the mouse as a model has been complicated by the finding that there are two homologous DAF genes in this species, and by the discovery of a rodent-specific membrane C3 convertase inhibitor known as Crry.25,26 Crry has both DAF and membrane cofactor protein (MCP) activities and is broadly expressed in mouse tissues.25,26 In vitro studies have established that both GPI-DAF and TM-DAF can function as effective C3 convertase inhibitors.16,20 It thus remains an intriguing question as to what are the relative roles of these three membrane C3 convertase inhibitors in the mouse. Although evidence from mRNA profiling has indicated that the GPI-DAF gene has a much wider tissue distribution,13–15 it is not yet known whether GPI-DAF and TM-DAF genes are expressed on the same cell types in tissues (such as the testis) where mRNAs for both genes were detected. Additionally, because erythrocytes are not amenable to mRNA analysis, the possible expression or lack of expression of the TM-DAF gene on mouse erythrocytes has not been ascertained. Furthermore, while the GPI-DAF gene knockout mouse survived and functioned normally,18 inactivation of the Crry gene proved to be lethal because of fetal susceptibility to maternal complement attack.19 These observations raised a number of issues including the relative expression of the three membrane inhibitors in the fetoplacental units and whether there is compensatory expression of the TM-DAF gene in the GPI-DAF gene knockout mice.

In this study, we have attempted to address some of these questions by examining the relative tissue expression patterns of the two DAF genes in wild-type and the GPI-DAF gene knockout mice, using several independently generated antibodies, with either dual or single specificity. The specificities of these antibodies were first evaluated on stably transfected HEK 293 and CHO cell lines expressing either the GPI-DAF or the TM-DAF cDNA. This experiment and previous studies confirmed that the polyclonal antibody, which was used in subsequent immunohistochemistry studies, was able to recognize both GPI-DAF and TM-DAF gene products. Similarly, two mAb, Riko-3 and 2C6.5 were confirmed to be cross-reactive with DAF proteins from both genes while one mAb Riko-4 recognized only GPI-DAF gene product (Fig. 120). Using all three monoclonal antibodies, we showed that DAF is present on wild-type mouse erythrocytes but no signal was detected on GPI-DAF gene knockout mouse erythrocytes. Given the known specificity of these antibodies, we can conclude that the TM-DAF gene is not expressed on erythrocytes of either the wild-type or the GPI-DAF gene knockout mice. It was observed in a previous study that DAF on mouse erythrocytes was resistant to PIPLC cleavage.17 Our finding in the present study suggests that the reason for this resistance must lie in a special feature of the GPI-anchor of mouse erythrocyte DAF rather than the presence of a transmembrane DAF derived from the TM-DAF gene.

Using two separate mAbs, we have determined that DAF is expressed on the majority of spleen B and T lymphocytes and macrophages. A sizeable percentage of CD3-positive spleen cells stained negative for DAF. The identity of these cells have not been determined but within the T-lymphocyte cell population, no difference in DAF staining exists between CD4+ and CD8+ cells (data not shown). It is interesting to observe that a much higher percentage of B cells than T cells were DAF-positive and the average level of DAF expression on B cells is also two- to three-fold higher than that on T cells (Fig. 3). It is not known if this differential expression on the two cell populations is related to DAF playing a more prominent role on B cells as a complement inhibitor or to its functioning in other capacity such as serving as a ligand for CD97. The demonstrated specific interaction between DAF and CD97,10–12 and the structural and expression characteristics of CD97 – being a seven transmembrane cell surface molecule that becomes rapidly expressed on T lymphocytes after cell activation – would be compatible with the hypothesis that DAF and CD97 constitute a pair of costimulatory molecules relevant to lymphocyte activation. Whatever the main functions of DAF in the spleen are, the fact that no signals were detected in the GPI-DAF gene knockout mouse spleen cells, either by FACS or immunohistochemistry, supports the conclusion that the TM-DAF gene is either not expressed or expressed at a level below the methods of detection. In a previous study using Northern blot analysis,14 mRNA message derived from the TM-DAF gene was detected in the mouse spleen. It is possible that there are alternatively spliced mRNA species from the GPI-DAF gene that cross-hybridized with a supposedly TM-DAF gene-specific probe.

By immunohistochemistry analysis, we have determined that the GPI-DAF and TM-DAF genes are differentially expressed in the mouse testis. Although both late-stage developing germ cells (spermatids) and mature sperm stained positive for DAF in the wild-type mouse testis, only mature sperm stained positive in the GPI-DAF gene knockout mouse testis (Fig. 5b,e). This finding indicated that the DAF protein present on spermatids is derived entirely from the GPI-DAF gene. Comparison of the staining intensity on mature sperm showed no obvious reduction in the GPI-DAF gene knockout mouse testis (Fig. 5b,e), suggesting that the GPI-DAF gene is probably not expressed on mature sperm. However, such a conclusion awaits confirmation through other independent means of evaluation. The role of the GPI-DAF gene in germ cell development is not known but does not appear to be indispensable since no male reproductive deficiency has been observed with the GPI-DAF gene knockout mice.18 Whether TM-DAF gene products on mature sperm play an essential role in male reproduction remains to be determined. There has been much speculation that complement regulatory proteins such as DAF may protect sperm from anti-sperm antibody-initiated complement damage in the female reproductive tract.27–29 Because GPI-anchored proteins are known to be shed easily from the membrane30 and because mature sperm have lost their cytoplasm and hence the ability to synthesize new proteins, it may be advantageous to have transmembrane rather than GPI-anchored DAF on the plasma membrane of sperm.

Another area of interest related to the study of human DAF is the demonstration that the protein is expressed prominently on human trophoblasts.22–24 This has led to the suggestion that DAF may play a role in protecting the developing embryos from adverse immunological reaction in early pregnancy.22–24 However, inactivation of the GPI-DAF gene in the mouse resulted in no reduction in fetal development or survival.18 In contrast, Crry-knockout mice were found to be highly susceptible to maternal complement attack and died during early development.19 We demonstrated in this study that although the GPI-DAF (but not the TM-DAF) gene is expressed in the decidua cells of the fetoplacental unit, neither the GPI-DAF gene nor the TM-DAF gene is expressed on mouse embryos at day-7·5, a developmental stage proved to be critical for the survival of Crry-deficient embryos.19 Crry has previously been shown to be expressed uniformly in the fetoplacental unit on cells of both fetal and maternal origin.19 Thus, the ability of GPI-DAF-deficient embryos to tolerate maternal complement attack does not reflect overlapping or compensating activity provided by the TM-DAF gene. Rather, adequate protection is conferred by Crry, which apparently is the sole C3 regulator expressed in early embryos in this species.

In summary, using several independently generated anti-mouse DAF antibodies and a strain of mouse which lacks the GPI-DAF gene expression, we have determined that TM-DAF gene expression in the mouse is restricted to mature sperm. In light of the highly limited expression of the TM-DAF gene and MCP,31,32 we conclude that potential compensatory activity in membrane C3 regulation in the apparently normal GPI-DAF gene knockout mice is most likely provided by Crry. Based on the present finding and results of GPI-DAF gene and Crry gene knockout mice,18,19 complement-mediated global tissue inflammation and damage might be expected in a mouse that is deficient in both GPI-DAF and Crry genes.

Acknowledgments

This work is supported by National Institutes of Health grant AI 44970. We are grateful to Dr Yang Luo for his generous help in the mouse embryo experiments.

References

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