The ITIM-bearing inhibitory receptor, CD72, has been reported to be expressed on B cells, T cells, NK cells, dendritic cells, and macrophages (13
). Here, we now demonstrate that CD72 is also expressed on human mast cells derived from CD34-positive peripheral blood and on human mast cell lines. This was demonstrated through the presence of mRNA for CD72, and by CD72 protein expression as detected by western blot and by FACS analysis (). Messenger RNA for CD72 was observed at all stages of development of the huMC cultures examined. However, there was a trend for reduced message in the more mature cultures. Thus, in mature mast cells (8 wk culture), CD72 protein expression was substantially lower than in the HMC 1.2 cells. Regardless, the surface expression as detected by FACS was comparable. A possible explanation for this is that, in HMC1.2 cells, the protein is partly retained in the cytosol (data not shown) due to defective regulation of translocation of CD72 protein to the cell surface following transcription in the HMC1.2 cells. Differences in CD72 protein expression between the huMCs and HMC1.2 cells may be reflective of the transformed nature of the latter cell type or may reflect an immature phenotype of the transformed cells.
Our data do differ from a previous study where the expression of CD72 by FACS analysis was not detected on human mast cells derived from umbilical cord blood (29
). However, in that report, the expression of mast cell function-associated antigen (MAFA), which is known to be expressed in mast cells (9
), was also not detected. The difference in CD72 expression between the two reports may thus reflect different sensitivities of the antibodies used or difference of the origins of the mast cell progenitors. Nevertheless, based on the data presented herewith in, CD72 can now be added to the list of inhibitory receptors which are documented to be expressed in mast cells.
The natural ligand for CD72 is recognized to be CD100, thus, an advantage of employing CD72 to down-regulate SCF-dependent mast cell function is that this can be achieved through interaction of CD72 with its natural ligand. Indeed, in this study we observed that the cellular responses induced by an anti-CD72 antibody could be mimicked by CD100. Expression of CD100 is reported in B cells, T cells and neuronal cells (30
), but to date has not been reported in mast cells. Our preliminary studies have also failed to detect CD100 expression in primary cultured human mast cells and in the HMC1.2 human mast cell line (data not shown). Regardless, it is possible that interaction between CD72-expressing mast cells and CD100-expressing immune cells may influence mast cell function, as is the case with the interaction among CD72-expressing B cells and CD100-expressing T cells (30
). Associations between mast cells and neurons have also been reported (31
). Thus, such events may also influence mast cell function via CD72 – CD100 interactions. Certainly, our data do provide supportive evidence that ligation of CD72 by CD100, or by means of an agonistic antibody, has the potential to modify KIT-mediated mast cell responses. In this respect, we observed that both rCD100 and an agonistic anti-CD72 antibody down-regulated the KIT dependent growth of human mast cells (), in addition to significantly reducing SCF-induced human mast cell chemotaxis, SCF-induced MCP-1 (CCL2) production (), and the SCF-enhancement of IgE-dependent degranulation ().
Cellular responses regulated by CD72 have been primarily investigated in B cells and B cell lines (13
). However, these studies have sometimes produced conflicting data (13
). Although the consensus of studies report that CD72 ligation positively regulates responses in B cells by reversing the inhibitory potential of CD72 (14
), other studies have revealed that CD72 ligation further increases its inhibitory potential (40
). Specifically, CD72 ligation has been reported to induce the proliferation of B cells (32
), and to positively regulate CD40-induced (14
) and antigen-mediated (36
) proliferation of B cells. Furthermore, CD72 ligation by an anti-CD72 antibody was reported to rescue B cell apoptosis mediated by B cell receptor (BCR) ligation and IgM hyper-crosslinking (38
). In contrast to these data, which imply that CD72 ligation reverses its inhibitory potential, CD72 expression in B cell line K46µm λ and incubation of splenic B cells with an anti-CD72 antibody resulted in down-modulation of BCR-mediated ERK activation and calcium mobilization (40
). These data imply that CD72 ligation promotes its inhibitory potential.
These apparently conflicting data have led to the conclusion that CD72 may regulate positive and negative signaling pathways for the regulation of B cell responses and that this may, in part, be related to the stages of B cell development (13
). Regardless, the ability of CD72 to regulate cellular responses is dependent on its phosphorylation status, hence its ability to recruit SHP-1, an interaction that is reported to be negatively influenced through its interaction with CD72-bound Grb2 (41
). It has been proposed that, in the scenario in which CD72 ligation reverses its inhibitory activity, rCD100 or the agonistic antibody results in dissociation of CD72 from the B cell signaling complex thus reversing BCR dependent phosphorylation of CD72. In contrast, in the scenario, in which CD72 ligation induces its inhibitory activity, the agonistic anti-CD72 antibody promotes association of CD72 with the receptor signaling-complex thus allowing its phosphorylation and recruitment of SHP-1 (40
In resting B cells, there appears to be minimal constitutive phosphorylation of CD72 (25
). However, in the huMCs, we observed that there was a slight but detectible constitutive phosphorylation of CD72 and its association with SHP-1 in the resting state (). Hence the possibility exists that CD72 may help regulate the basal activation state of the human mast cells. Our data, furthermore, demonstrate that in these cells, ligation of CD72 with either rCD100 or the agonistic antibody BU40 concurrently with KIT activation, results in the activation of necessary events which allow CD72 to inhibit Kit-mediated signaling. Thus, in the case of mast cells, the mode of the responses elicited by rCD100 and BU40 would appear similar to that reported in the splenic B cells (40
), which is suggestive of permissive phosphorylation of CD72 by KIT following CD72 ligation. This conclusion was further supported by the enhancement of tyrosine phosphorylation of CD72 observed in the CD72-immunoprecipitates from mast cells incubated with BU40 or rCD100 and triggered through KIT ().
Unlike the BCR, KIT possesses inherent catalytic activity which is increased upon SCF-induced KIT dimerization. An increase in CD72 phosphorylation was not observed in cells incubated with rCD100 or BU40 in the absence of KIT activation (). It is likely that the inducible phosphorylation of CD72 observed in the human mast cells is directly due to phosphorylation by KIT, contrary to the case in immature B cells. Our data further demonstrated that there was a significant increase in the association of SHP-1 with the tyrosine phosphorylated CD72 (). SHP-1 has been demonstrated to down-regulate KIT signals in vivo (42
) and, indeed, we observed that the phosphorylation of SHP-1, which is known to increase its phosphatase activity (26
), was elevated in the huMCs () and HMC1.2 cells () following CD72 ligation. In addition to CD72, SHP-1 has also been shown to be associated with other inhibitory receptors which down-regulate KIT-mediated responses in mast cells (12
). Thus, we can conclude that the ability of ligated CD72 to inhibit KIT-mediated responses in mast cells is linked to its ability to recruit SHP-1 following its phophorylation.
SHP-1 dephosphorylates regulatory tyrosine residues on critical proteins which participate in signaling cascades initiated by multiple receptors including KIT (6
). We observed that CD72 phosphorylation led to the suppression of the phosphorylation of KIT, SFKs, and ERK induced by SCF challenge (), but not of AKT or Stat3. Both KIT and SFKs are known to be directly dephosphorylated by interactions with SHP-1 (43
). Therefore, it is likely that the down-regulated phosphorylation of KIT and SFKs induced by costmulation of KIT with CD72 in mast cells was a consequence of the formation of the CD72 – SHP-1 complex. It is unclear whether SHP-1 can directly regulate the activation of ERK (46
). Therefore there are two possible explanations for the observed ERK dephosphorylation observed in the human mast cells in response to co-activation of KIT and CD72: (i) The suppressed activation of ERK by CD72 stimulation was mediated directly by the CD72 – SHP-1 complex or; (ii) the suppressed ERK phosphorylation was indirect due to the down-regulation of SFKs activity, as ERK phosphorylation is known to be regulated by SFKs (48
Regardless of whether direct or indirect, the down-regulation of the KIT-dependent phosphorylation of KIT, SFKs and ERK by the interaction of SHP-1 with phosphorylated CD72 would certainly account for the ability of ligated CD72 to down-regulate KIT-mediated responses in human mast cells. In this respect, in mast cells, the SFKs, Lyn and Fyn, play important roles in KIT-mediated proliferation and chemotaxis (49
) and ERK participates in the process of KIT-mediated proliferation and MCP-1 production (50
). The downregulation of SCF-induced phosphorylation of SFKs by CD72 would also account for the observed inhibition of SCF-enhanced degraulation following CD72 ligation (). The inability of ligated CD72 to decrease the degranulation response induced by FcεRI aggregation alone again points to the requirement of direct phosphorylation of CD72 by Kit to induced inhibitory responses. Taken together, from the above conclusions, we can now put together a model of how CD72 may regulate KIT-mediated human mast cell function ().
Proposed model of the effects of CD72 – CD100 system on human mast cells. SFKs; Src family kinases, ERKs; extracellular-regulated kinases.
In summary, we have presented data which demonstrates that the ITIM-containing inhibitory receptor CD72 is expressed in human mast cells and mast cell lines. When ligated either by its natural ligand CD100 or by an anti-CD72 antibody, concurrently with activated KIT, CD72 becomes phosphorylated thereby recruiting SHP-1 resulting in dephosphorylation of critical signaling molecules resulting in down-regulation of KIT-mediated mast cell activation. The ability of ligated CD72 to also down-regulate the growth of tumor mast cells provides evidence of the potential application of CD72 in mast cell disorders such as mastocytosis.