Involvement of CD100, a lymphocyte semaphorin, in the activation of the human immune system via CD72: implications for the regulation of immune and inflammatory responses

Isao Ishida1, Atsushi Kumanogoh1, Kazuhiro Suzuki1, Shiro Akahani2, Kazuhiro Noda2 and Hitoshi Kikutani1

1 Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan 2 Department of Otolaryngology and Sensory Organ Surgery, Osaka University Graduate School of Medicine, 2-2 Yamada-Oka, Suita, Osaka 565-0871, Japan

The first two authors contributed equally to this work
Correspondence to: H. Kikutani; E-mail: kikutani{at}ragtime.biken.osaka-u.ac.jp
Transmitting editor: K. Inaba


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD100/Sema4D belongs to the semaphorin family, factors known to act as repulsive cues for axons during neuronal development. Mouse CD100 plays a crucial role in both humoral and cellular immunity through ligation of the lymphocyte receptor, CD72. It remains controversial, however, whether human CD100 can function through human CD72 in a manner similar to mouse CD100. To determine the function of human CD100, we generated a recombinant soluble human CD100 protein comprised of the extracellular region of human CD100 fused to the human IgG1 Fc region (hCD100–Fc). hCD100–Fc specifically binds to cells expressing human CD72. As observed previously in the mouse, hCD100–Fc induces the tyrosine dephosphorylation of human CD72, leading to the dissociation of SHP-1 from the CD72 cytoplasmic tail. Consistent with findings for mouse CD100, hCD100–Fc exerts a co-stimulatory effect on B cells and dendritic cells that are stimulated with anti-CD40 mAb. Furthermore, both hCD100–Fc and anti-human CD72 agonistic mAb induce the production of the pro-inflammatory cytokines tumor necrosis factor-{alpha}, IL-6 and IL-8, even in the absence of anti-CD40 mAb. Collectively, our findings not only demonstrate that human CD100, interacting with human CD72, can function as a ligand in a manner similar to mouse CD100, but also suggest the involvement of human CD100 in inflammatory responses.

Keywords: CD40, plexin-B1, SHP-1


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD100/Sema4D, a 150-kDa transmembrane glycoprotein, is a member of the semaphorin family (1,2). Several members of this family play crucial roles in axonal guidance, functioning as chemorepulsive factors acting during neuronal development (35). Recent evidence suggests that several semaphorins, including the class IV semaphorins, CD100 and Sema4A, and the viral semaphorins, A39R and AHVsema, aid in the regulation of immune responses (2,610). CD100 is the first semaphorin shown to be expressed in the immune system (1,11). CD100 mRNA is found at high levels within both lymphoid organs, such as the spleen, thymus and lymph nodes, and non-lymphoid organs, including the brain and kidney (12,13). While CD100 expression is abundant on the surface of resting T cells, it is relatively weak on resting B cells and professional antigen-presenting cells, such as dendritic cells (DC), although it is up-regulated upon activation (2,6,7).

Early descriptions of CD100 using anti-human CD100 mAb suggested a possible function as a receptor capable of signaling through the cytoplasmic domain (1,11,14). Antibody-mediated cross-linking of human CD100 provides proliferative signals to human peripheral blood T cells in the presence of submitogenic doses of either anti-CD3 or anti-CD2 mAb. In addition, tyrosine phosphatases, including CD45, are associated with CD100 in human T cells (15). Furthermore, serine/threonine kinase activity is reportedly associated with the cytoplasmic domain of human CD100 in T cells and NK cells (16).

Following the revelation of CD100 as a member of the semaphorin family through molecular cloning (12), cumulative evidence suggests that CD100 functions as a ligand, as other members of the semaphorin family do. The addition of mouse CD100-expressing cells or soluble mouse CD100 to B cells significantly enhances CD40-induced proliferation and differentiation (6,17,18). Administration of soluble mouse CD100 protein also accelerates in vivo antibody responses against T cell-dependent antigens (6). Soluble mouse CD100 enhances CD40-induced DC maturation, as measured by the up-regulation of co-stimulatory molecules and IL-12 production (7). Furthermore, defects in T cell priming in CD100-deficient mice can be ameliorated by the administration of soluble mouse CD100 (19). Concerning CD100 function in the human system, human CD100-expressing transfectants promote B cell aggregation and survival in vitro while concurrently inducing the shedding of CD23 from the surface of B cells (12). Delaire et al. have also shown that soluble human CD100 inhibits both the spontaneous and chemokine-induced migration of immune cells (20). Together, these findings indicate that CD100 functions as a ligand of specific receptors in both the mouse and human systems, although the biological functions of human CD100 remain unclear.

Mouse CD100 utilizes CD72 as a receptor in lymphoid tissues, although plexin-B1 may fill this role in non-lymphoid tissues (6,21). CD100-deficient mice have multiple defects in lymphoid tissues (19), but not within the tissues in which plexin-B1 is expressed abundantly (22), suggesting that the interaction between CD100 and CD72 in the immune system is not redundant. CD72 is a 45-kDa type II transmembrane protein belonging to the C-type lectin family (2325). CD72 functions as a negative regulator of B cell responses by recruiting SHP-1, a tyrosine phosphatase, to its immunoreceptor tyrosine-based inhibitory motif (26). Both anti-mouse CD72 mAb and soluble mouse CD100 protein can induce the dephosphorylation of mouse CD72, facilitating the dissociation of SHP-1 from mouse CD72 (6,27). Thus, CD100 stimulation appears to turn off the negative signaling mediated by CD72, resulting in enhancing B cell responses (2). Supporting this possibility, the immunological phenotype of CD100-deficient mice is almost the opposite to that of CD72-deficient mice (19,28). In CD100-deficient B cells, CD72 is constitutively tyrosine phosphorylated, leading to a constant association with SHP-1 (19). Therefore, observations in the mouse system indicate that the CD100–CD72 interaction is critical in the regulation of immune responses. The precise molecular mechanism by which human CD100 exerts its activity, however, is not known. It remains controversial if human CD100 utilizes CD72 as its receptor.

In this study, we demonstrate that human CD100 specifically binds to human CD72. In a manner similar to mouse CD100, human CD100 stimulation enhances the proliferation of B cells and maturation of DC in the presence of CD40 stimulation. Furthermore, we demonstrate that, even in the absence of CD40 stimulation, both soluble human CD100 and anti-human CD72 mAb can induce the production of pro-inflammatory cytokines by monocytes, suggesting a role for the CD100–CD72 interaction in generalized inflammatory responses.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of soluble human CD100 proteins
A truncated form of human CD100 cDNA was prepared from the full-length human CD100 cDNA. This cDNA was isolated from human peripheral blood mononuclear cells (PBMC) by PCR using a pair of oligonucleotide primers, a sense sequence containing a SalI site (5'-ATGTCGACTCGCCTC TACCTGATGAGGATGTGCACCCCCATTA-3') and an antisense sequence including a BglII site (5'-ATAGATCTTACT TACTTTGCTTAAGATACATGGTTTTCTCCGA-3'). The resulting SalI–BglII fragments replaced the SalI–BamHI DNA fragment of the pEFBos human IgG1 Fc cassette (29), generating the human CD100–Fc protein. To produce soluble human CD100 protein, we established stable P3U1 plasmacytoma transformants carrying the expression plasmids by electroporation. Briefly, aliquots of 107 cells were transfected by electroporation with 30 µg pEFBos hCD100–Fc digested with HindIII and 3 µg pMC1neo digested with BamHI. After 10 days of selection in RPMI 1640 medium containing 10% FCS and 0.3 mg/ml G418, G418-resistant colonies were isolated and cloned. hCD100–Fc protein was purified from culture supernatants by Protein A–Sepharose isolation (Amersham Biosciences, Uppsala, Sweden).

Cells
Informed consent was obtained from all human subjects; the study was performed according to the institutional guidelines. PBMC were isolated by density-gradient centrifugation of heparinized blood obtained from healthy donors using Ficoll-Hypaque (Amersham Biosciences). Monocytes were purified from PBMC through the depletion of T cells and B cells using anti-CD3 and anti-CD19 Dynabeads (Dynal, Oslo, Norway) respectively, yielding a resulting purity of >95% CD11b+ cells. Monocyte-derived DC were generated from PBMC by 6 days culture of granulocyte macrophage colony stimulating factor and IL-4, as previously described (30,31) (>95% CD11c+ cell purity). Tonsils were obtained by routine tonsillectomies; residing mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation. Small resting B cells were obtained on a Percoll (Amersham Biosciences) density gradient. The cell population at the interface between 50 and 55% was recovered, resulting in >90% CD19+ cells.

Cytokine assays
To measure tumor necrosis factor (TNF)-{alpha}, IL-6 and IL-8 production by monocytes, 1 x 105 cells were stimulated for 48 h with or without various concentrations of anti-human CD40 mAb (5C3; mouse IgG1; BD PharMingen, San Diego, CA), hCD100–Fc, anti-human CD72 mAb (BU40; mouse IgG2a, Southern Biotechnology Associates, Birmingham, AL) or CD40–Fc in flat-bottomed 96-well microtiter plates. To measure IL-12 production by monocyte-derived DC, cells were stimulated with or without various concentrations of anti-human CD40 mAb, hCD100–Fc, anti-human CD72 mAb, human Ig fractions (Chemicon, Temecula, CA) or mouse IgG2a. Following 48 h of stimulation, cytokine levels in culture supernatants were measured using ELISA kits (R & D Systems, Minneapolis, MN).

B cell proliferation assays
Tonsillar B cells (1 x 105/ml) were stimulated for 72 h with or without anti-human CD40 mAb (20 µg/ml), hCD100–Fc (0.8, 4 and 20 µg/ml), anti-human CD72 mAb (0.8, 4 and 20 µg/ml), human Ig (20 µg/ml) or isotype-matched controls (20 µg/ml) in flat-bottomed 96-well microtiter plates. Cells were pulsed with 2 µCi [3H]thymidine during the last 16 h.

Transfectants
The full-length human cDNA encoding CD72 was transfected into CHO cells using Lipofectamine Plus 2000 (Invitrogen-Life Technologies, Carlsbad, CA) to create stable CD72-CHO cells. Transfectants expressing human CD72 were selected by flow cytometry following staining with FITC-conjugated anti-human CD72 mAb (J4-117; BD PharMingen), then cloned.

Flow cytometric analysis
For CD72-CHO cells, 1 x 106 cells were incubated with either biotinylated hCD100–Fc or biotinylated human IgG1, followed by allophycocyanin (APC)-conjugated streptavidin and FITC-conjugated anti-human CD72 mAb. For B cells, 1 x 106 cells were stained with biotinylated hCD100–Fc in the presence or absence of excess of anti-human CD72 mAb or isotype-matched controls, then incubated with APC-conjugated streptavidin in the presence of human Ig to block Fc{gamma} receptors. For monocytes, 1 x 106 cells were stained with phycoerythrin-conjugated anti-human CD40 mAb (mAb89; Immunotech, Marseille, France) and biotinylated anti-human CD72 mAb (J3.109; Immunotech), followed by FITC-conjugated streptavidin. For monocyte-derived DC, 1 x 106 cells were stained with FITC-conjugated anti-human CD40 mAb (5C3) and biotinylated anti-human CD72 mAb (J3.109), followed by FITC-conjugated streptavidin in the presence of human Ig to block Fc{gamma} receptors. Cells were analyzed by flow cytometry. Data analysis was performed using Flow Jo software (Treestar, San Carlos, CA).

Immunoprecipitation and Western blotting
Cells were lysed in 1% Nonidet P-40, containing 150 mM NaCl, 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 1 mM Na3VO4, 0.5 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin and Complete protease inhibitor cocktails (Roche, Basel, Switzerland). Following preclearing with Protein A–Sepharose beads, cell lysates were incubated for 3 h with Protein A–Sepharose beads plus either anti-CD72 (H-96; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-SHP-1 (C-19; Santa Cruz Biotechnology) at 4°C. After extensive washing, immunoprecipitates were subjected to SDS–PAGE and then transferred to nitrocellulose membranes. Membranes were incubated with anti-CD72 (H-96), anti-SHP-1 (C-19) or anti-phosphotyrosine antibodies (4G10; Upstate Biotechnology, Lake Placid, NY). Blots were developed by enhanced chemiluminescence (ECL) following the manufacturer’s protocol (Amersham Biosciences).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Human CD100 binds to human CD72
To investigate the function of human CD100, we prepared a soluble human CD100, hCD100–Fc, consisting of the putative extracellular region of human CD100 fused to the human IgG1 Fc region. A band of ~150 kDa was observed for hCD100–Fc following reduction. In addition, a larger band appeared under non-reducing conditions (Fig. 1A). This larger band may represent a homodimer of hCD100–Fc because both the Fc portion of IgG1 and an extracellular region of CD100 are known to form homodimers through disulfide bonds (11). We established human CD72-expressing CHO cell transfectants (CD72-CHO) (Fig. 1B). Biotinylated hCD100–Fc specifically bound to CD72-CHO, but not to control CHO, cells transfected with a neomycin resistance plasmid alone (CHOneo). As CD72 was originally identified as a B cell-surface antigen (32), we next examined the binding of hCD100–Fc to human B cells; hCD100–Fc also bound to human tonsillar B cells (Fig. 1C). In addition, anti-human CD72 mAb, but not isotype-matched controls, could block hCD100–Fc binding to human tonsillar B cells, confirming that human CD100 binds to human CD72, as reported for mouse CD100 and CD72 (6).



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Fig. 1. Human CD72 is a receptor for human CD100. (A) SDS–PAGE analysis of purified hCD100–Fc. Purified hCD100–Fc (3 µg) was separated by gradient PAGE (left: 4–20%; right: 3–8%) in the presence of 0.1% SDS under either reducing [2-mercaptoethanol (+)] or non-reducing [2-mercaptoethanol (–)] conditions and then visualized by silver staining. Molecular weight markers (kDa) are shown at the left. (B, upper) CD72-CHO or CHOneo cells were stained with FITC-conjugated anti-human CD72mAb (solid line) or FITC-conjugated isotype controls (dotted line). (B, lower) CD72-CHO cells were stained with biotinylated hCD100–Fc (solid line) or biotinylated human IgGs (dotted line), followed by incubation with APC-conjugated streptavidin. Samples were analyzed by flow cytometry. (C) Tonsillar B cells were stained with biotinylated hCD100–Fc (2 µg/ml, bold line), biotinylated hCD100–Fc in the presence of anti-human CD72 mAb (100 µg/ml, thin line), isotype-matched mouse IgG2a (100 µg/ml, bold dotted line) or biotinylated human IgGs (2 µg/ml, dotted line) followed by APC-conjugated streptavidin.

 
hCD100–Fc induces tyrosine dephosphorylation of human CD72 and the dissociation of SHP-1 from human CD72
Mouse CD100–Fc induces CD72 tyrosine dephosphorylation and SHP-1 dissociation from CD72 in COS7 cells transfected with a mouse CD72 cDNA (6). When human CD72 cDNA was transiently transfected into COS7 cells, its protein product was constitutively tyrosine phosphorylated and associated with SHP-1 (Fig. 2), as previously observed for mouse CD72. hCD100–Fc also induced the tyrosine dephosphorylation of human CD72 and dissociation of SHP-1. This finding suggests that human CD100 evokes similar signaling pathways through human CD72 as the cognate mouse molecules.



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Fig. 2. Human CD100 induces human CD72 tyrosine dephosphorylation and the dissociation of SHP-1 from CD72. COS7 cells were transfected with human CD72 cDNA by lipofection. Two days after transfection, cells were stimulated with or without hCD100–Fc for 10 min. CD72 or SHP-1 were immunoprecipitated from cell lysates (1% NP-40) with anti-CD72 or anti-SHP-1 respectively and visualized with anti-phosphotyrosine, anti-SHP-1 or anti-CD72.

 
Human CD100 enhances CD40-induced proliferation of human tonsillar B cells
Mouse CD100–Fc synergistically enhances CD40-induced B cell proliferation (6). We next examined the efficiency of human tonsillar B cell stimulation by hCD100–Fc. Although mouse CD100–Fc alone does not affect B cells, hCD100–Fc induced weak, but substantial, proliferation of B cells in the absence of anti-CD40 stimulation. In addition, hCD100–Fc enhanced CD40-induced B cell proliferative responses, as seen for mouse CD100–Fc (Fig. 3). Agonistic anti-human CD72 mAb also both induced B cell proliferation even in the absence of CD40 signaling and enhanced CD40-induced proliferation. These results suggest that human CD100 and anti-human CD72 mAb exert similar effects on human B cells, paralleling the activities of mouse CD100 and anti-mouse CD72 mAb. It is noteworthy that anti-human CD72 mAb is a more potent stimulant for B cells in comparison to hCD100–Fc.



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Fig. 3. CD100 enhances CD40-mediated B cell responses. Small resting B cells were cultured for 72 h in the presence of hCD100–Fc, anti-human CD40 mAb (5C3; mouse IgG1, 20 µg/ml) or anti-human CD72 mAb (BU40; mouse IgG2a). Human IgG (20 µg/ml), mouse IgG1 (20 µg/ml) and mouse IgG2a (20 µg/ml) were used for the respective controls. Cells were pulsed with 2 µCi [3H]thymidine for the last 16 h of incubation. Data are the mean ± SD of triplicate wells. The results shown are representative of three independent experiments.

 
Human CD100 induces IL-12 production by DC
CD72 is expressed on the surface of mouse splenic and bone marrow-derived DC, playing a role in the activation and maturation of DC (7). We first examined the expression of CD72 on monocytes and monocyte-derived DC after culture with GM-CSF plus IL-4. As shown in Fig. 4, the expression of CD40 and CD72 was detected on both monocytes and monocyte-derived DC. We next examined the effect of human CD100 on IL-12 production by monocyte-derived DC. hCD100–Fc enhanced CD40-induced IL-12 production (Fig. 5). The synergy between hCD100–Fc and anti-CD40 mAb stimuli was particularly evident when cells were stimulated with suboptimal doses of anti-CD40 mAb. Anti-CD72 mAb also induced significant amounts of IL-12 production by DC, indicating that human CD100 also stimulates DC through CD72.



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Fig. 4. Expression of CD40 and CD72 on the cell surface of monocytes and monocyte-derived DC. Monocytes (A) or monocyte-derived DC (B) from healthy adult donors were stained with either FITC-conjugated anti-human CD40 mAb (solid line) or FITC-conjugated isotype controls (dotted line), with either biotinylated anti-human CD72 mAb (solid line) or biotinylated isotype controls (dotted line), followed by APC-conjugated streptavidin and then analyzed by flow cytometry.

 


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Fig. 5. CD100 enhances IL-12 production by human monocyte-derived DC. (A) Monocyte-derived DC were stimulated with either hCD100–Fc (0.8, 4 and 20 µg/ml) or control human IgG (20 µg/ml) in the presence of varying concentrations of anti-human CD40 mAb. (B) Monocyte-derived DC were stimulated for 48 h with anti-human CD72 mAb (0.8, 4 and 20 µg/ml) or control isotype-matched mouse IgG2a (20 µg/ml) in the presence of varying concentrations of anti-human CD40 mAb. Supernatants were assayed for IL-12 p40 by ELISA. Data are the mean ± SD of triplicate wells. The results shown are representative of five independent experiments.

 
Human CD100 induces pro-inflammatory cytokine production by monocytes
The activity of human CD100 to inhibit human blood monocytic cell migration suggests the involvement of human CD100 in monocyte-mediated immune responses (20). Monocytes play a crucial role in inflammatory responses through the production of various pro-inflammatory cytokines, including TNF-{alpha}, IL-6 and IL-8. We examined the effects of hCD100–Fc on cytokine production by human blood monocytes. CD100 or anti-CD72 mAb alone could induce cytokine production (Fig. 6). Neither hCD100–Fc nor anti-human CD72 mAb altered CD40-induced cytokine production (data not shown), which is different from that seen in B cells and DC.



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Fig. 6. Enhancement of pro-inflammatory cytokine production in human monocytes. Monocytes from healthy adult donors were cultured with or without CD40–Fc (20 µg/ml) (39), hCD100–Fc (0.8, 4 and 20 µg/ml), anti-human CD72 mAb (0.8, 4 and 20 µg/ml) or anti-hCD40 mAb (20 µg/ml) for 48 h. TNF-{alpha} (A), IL-6 (B) and IL-8 (C) in the culture supernatants were measured by ELISA. Data are the mean ± SD of triplicate wells. The results shown are representative of five independent experiments.

 
Expression of plexin-B1 in immune cells
It has been recently reported that human plexin-B1 sustains proliferation of chronic lymphocytic leukemia cells and B1 B cells, both of which express CD100 (33), suggesting a possible immunological role of CD100–plexin-B1 interactions. Therefore, we examined the expression of human plexin-B1 in human CD100-responding cells including B cells, DC and monocytes, all of which express CD72 as shown in Figs 1 and 4. The expression of human plexin-B1 could not be detected in these cells, although it was slightly detected in activated T cells (Fig. 7). These expression profiles of human plexin-B1 were consistent with the previous report (33). Collectively, these findings suggest that human CD100 exerts immunological activities on B cells, DC and monocytes as a ligand, acting through human CD72.



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Fig. 7. Expression of plexin-B1. RT-PCR for expression of human plexin-B1. RNA was isolated from the human kidney, B cells, DC, monocytes and T cells before and after phytohemagglutinin (5 µg/ml) stimulation, and then treated with DNase I to exclude genomic DNA. PCR was performed as described previously (33).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we demonstrate that human CD100, that interacts with human CD72, has similar biological properties to mouse CD100. Human CD100 specifically binds to human CD72-expressing cells. Human CD100 enhances CD40-mediated B cell responses. The binding of human CD100 to human CD72 induces CD72 tyrosine dephosphorylation and dissociation of SHP-1. Human CD100 is also involved in maturation of bone marrow-derived DC. Together, these results indicate that human CD100 exerts similar effects as mouse CD100 (2). In addition, we also determined that human CD72, but not plexin-B1, was expressed in B cells, DC and monocytes, on which hCD100–Fc was effective. Collectively, our findings suggest that human CD100 functions through human CD72 in a manner similar to mouse CD100.

In vivo administration of soluble recombinant mouse CD100 to mice promotes antigen-specific antibody production and the generation of antigen-specific T cells (6,18,19). Stimulation of antigen-pulsed DC with both recombinant mouse CD100 and anti-CD40 mAb significantly augments their in vivo immunogenicity, leading to the enhanced generation of antigen-specific T cells and memory T cells (7). Blockade of CD100 inhibits T cell priming; CD100-deficient mice resist the development of experimental autoimmune encephalomyelitis, due to impaired generation of antigen-specific T cells (7). These previous findings strongly suggest that CD100 may be a valuable potential target for both the excitation of immunity against infection and tumors and the intervention in autoimmune diseases. The present study with human CD100 further substantiates the importance of this molecule in immunity, demonstrating conserved action throughout these two mammalian models.

There are, however, notable differences between the effects of human and mouse CD100; the effects of mouse CD100 can be observed only in the presence of CD40 stimulation (6,7), while human CD100 exerted an effect on monocytes even in the absence of an additional signal. The reason for this is that the human cells used in this study may be stimulated by some components of FCS. These signals may normally be suppressed by CD72, which is relieved by the addition of soluble CD100 or anti-CD72 mAb. It is also worthy of note that the effects of anti-CD72 mAb appear to be more potent than those of human CD100. This is probably because the affinity between anti-CD72 mAb and CD72 is higher than that between CD100 and CD72. In addition, it may be possible that conformational change induced by anti-CD72 mAb has a stronger effect on CD72 signals than that induced by CD100. Even in the absence of CD40, either hCD100–Fc or anti-CD72 mAb induced cytokine production by monocytes. This observation suggests that CD72 may transmit not only negative, but also positive signals. Anti-CD72 mAb alone induces the tyrosine phosphorylation of several signaling molecules, including CD19,Syk, Lyn and Btk (34,35). In addition to SHP-1, CD72 also interacts with BLNK and Grb2 (36), both thought to function as positive regulators. Thus, positive signaling of CD72 may predominate over negative signals in monocytes. Further studies will be required to determine if the stimulatory effects of CD100 can be explained by positive signaling through CD72.

Although cumulative evidence indicates that CD100 can function as a ligand, acting through its specific receptors, CD72 and plexin-B1 (2,21), it has been suggested that human CD100 can also function as a receptor. It has been reported that antibody-mediated cross-linking of human CD100 results in the proliferation of T cells in the presence of suboptimal doses of anti-CD3 mAb (11). Furthermore, it has been reported recently that human plexin-B1-expressing transfectants sustain the proliferation of normal and leukemic CD5+ cells, both of which express human CD100 (33), suggesting that human plexin-B1 can function as a ligand for human CD100. However, we have not observed the effects of CD72 as a ligand for CD100 as far as we examined (data not shown), suggesting that CD72 can function only as a receptor for CD100 in the CD100–CD72 interactions. Further studies will be required to evaluate the role of CD100–CD72 interactions in human systems.

We demonstrated that human CD100 stimulation induces the production of the pro-inflammatory cytokines, TNF-{alpha}, IL-6 and IL-8, by monocytes. Delaire et al. recently reported that soluble CD100 inhibits spontaneous and MCP-3-induced monocytic cell migration (20). Therefore, CD100 may exert an anti-inflammatory activity by inhibiting the transmigration of monocytes from the blood to inflammatory sites. Although the physiological significance of the contrary effects of CD100 on monocytes remains poorly understood, CD100 may regulate inflammatory responses in both a positive and negative manner. A class III semaphorin, Sema3A, inhibits monocyte migration through a receptor that may also bind CD100 (20). While either neuropilin-1, a receptor for Sema3A, or plexin-B1 is not expressed in monocytes (20,33), the possibility that CD100 and Sema3A may inhibit monocyte migration through CD72 remains unclear.

Previous studies have also suggested viral semaphorins, including A39R and AHVsema, as stimulants for human monocytes. These virally encoded semaphorins induce IL-6 and IL-8 production, enhancing the expression of CD54 by monocytes (10). In addition, CD108/Sema7A, a human homologue of AHVsema, induces monocyte pro-inflammatory cytokine production and chemotaxis (37). These findings indicate the critical involvement of semaphorins in inflammatory immune responses. Semaphorin molecules are phylogenetically conserved proteins from invertebrates to vertebrates. While the majority of studies examining the immunoregulatory semaphorins have been focused on the adaptive immune system using B cells, T cells and DC (38), future studies hope to clarify the role of immunoregulatory semaphorins in conserved innate immunity pathways common to both vertebrates and invertebrates.


    Acknowledgements
 
We would like to thank Ms Kyoko Kubota for her excellent administrative assistance. This work was supported by research grants from the Ministry of Education, Culture, Science and Technology of Japan to A. K. and H. K.


    Abbreviations
 
APC—allophycocyanin

DC—dendritic cell

hCD100–Fc—human CD100 fused to human immunoglobulin G1 Fc region

TNF—tumor necrosis factor

PBMC—peripheral blood mononuclear cell


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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