BACKGROUND
members of the transforming growth factor β  superfamily play central roles in controlling cellular proliferation, differentiation, migration and apoptosis  <cit> .
these cytokines can be divided into three subgroups: tgf-β, the activins/inhibins, and the bone morphogenetic proteins , of which the latter constitute the largest family.
bmps are 30– <dig> kda hetero- or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation  <cit> .
several studies have demonstrated an essential role of these proteins during embryogenesis, and more recently, also in adult tissues  <cit> .
tgf-β has been intensively studied in normal and malignant haematopoietic cells and is one of the most potent endogenous negative regulators known to date.
<cit> .
in contrast, the effect of bmps in the immune system has not been widely investigated.
in that respect, bmp-  <dig>  - <dig> and - <dig> have been found to control differentiation of hematopoietic stem cells  <cit>  and early t cell development  <cit> .
bmp- <dig> has been reported to reduce the number of cobblestone-area-forming cells of normal human haematopoietic cells  <cit> .
furthermore, bmp- <dig>  - <dig>   <dig> and - <dig> had an antiproliferative and a proapoptotic effect on multiple myeloma cells  <cit> .
in addition, by gene expression profiling, bmp- <dig> significantly increased the predictive value for a multi-gene signature test and was associated with a poor outcome in diffuse large b cell lymphomas   <cit> .
bmp- <dig>  like the other bmp members, signals through ligation and heterodimerzation of bmp type i  and type ii serine-threonine kinase receptors, which subsequently propagates the signal downstream by phosphorylating smad proteins.
bmp- <dig> can signal through the ligation of the type i receptors act-ria, bmp-ria, and bmp-rib and the type ii receptors bmp-rii, act-riia and act-riib, which lead to the phosphorylation of the receptor smads .
the r- smads then form complexes with the co-smad  and are translocated into the nucleus where they exert gene regulation  <cit> .
given the reported role of bmp- <dig> in b-cell malignancies and haematopoietic progenitor cells, we wanted to explore its potential role in normal human b cells.
we studied the effects of bmp- <dig> on proliferation and apoptosis on resting and stimulated b cells.
furthermore, the expression of bmp receptors and bmp- <dig> induced activation of the smad signalling pathway with subsequent regulation of the target genes id1–id <dig>  were resolved.
finally, we investigated whether b cells also were capable of producing bmp- <dig>
RESULTS
bmp- <dig> inhibits anti-igm induced proliferation of human b cells
the effects of bmp- <dig> on normal and neoplastic hematopoietic cells prompted us to investigate the effects of bmp- <dig> on normal human b cells.
all experiments in this study were performed under serum-free conditions as fcs has been shown to interfere with bmp-signalling  <cit> .
to study the effect of bmp- <dig> on proliferation, b-cells from healthy volunteers were stimulated with anti-igm and/or cd40l in the presence or absence of bmp- <dig> for three days.
we found that bmp- <dig> led to a 35% mean reduction of anti-igm- induced dna synthesis .
similar results were obtained for b cells treated with anti-igm and cd40l .
the bmp-6-induced inhibition of proliferation was dose-dependent in both peripheral b cells  and the burkitt lymphoma cell line ramos .
the bmp- <dig> effects could be reversed by addition of the extracellular inhibitor noggin .
similarly, a combination of the soluble bmp receptors bmp-rib-fc and bmp-rii-fc also neutralized the effects of bmp- <dig> .
next, we wanted to test whether bmp- <dig> had different effect on naïve and memory b cells.
naïve  and memory  b cells were isolated from peripheral blood by cell sorting of immunobead-isolated cd19+ b cells  <cit> , and tested for their capacity to proliferate in the presence of bmp- <dig>  however, bmp- <dig> inhibited anti-igm induced dna synthesis in the two subpopulations to a similar extent, with a mean reduction of dna-synthesis of 45%  for naïve b cells and 48%  for memory b cells .
bmp- <dig> induces cell death in human memory b cells and ramos cells
next, we wanted to establish whether bmp- <dig> also could affect the viability of normal b cells.
cell viability was determined by propidium iodide  staining after culture with or without bmp- <dig> for  <dig> hours.
interestingly, bmp- <dig> showed a small, but reproducible mean increase of cell death from  <dig> to 23%  in anti-igm stimulated cd27+ memory b cells.
furthermore, ramos cells showed a mean increase in cell death from  <dig> to 50%  after bmp- <dig> treatment.
in contrast, cell death of total cd19+ cells  or cd27-igg- naïve b cells was not significantly affected .
human b cells express bmp- <dig> receptors
detailed knowledge regarding expression of different bmp receptors in b cells is currently not available.
to further elucidate the role of bmps in human b cells, we performed western blot analysis for type i and type ii bmp receptors.
this analysis revealed that the type i receptors act-ria, bmp-rib and the type ii receptors bmp-rii and act-riib are expressed on resting human b-cells .
ramos cells expressed the type i receptors act-ria, weakly bmp-rib and the type ii receptor bmp-rii, but more weakly than normal b cells .
hl <dig> cells were used for comparison and weakly expressed act-ria and bmp-rii.
taken together, these data show that normal human b cells and ramos cells express a set of bmp receptors, previously shown to bind bmp- <dig>  <cit> .
bmp- <dig> induces phosphorylation of smad1/5/8
upon ligand binding, the type ii receptor transphosphorylates and activates the type i receptor.
type i receptors can signal via several pathways.
we examined the effect of bmp- <dig> on smad phosphorylation, as the activation of smad is considered to be a major signalling pathway for bmps  <cit> .
b cells were cultured in serum-free media over night and then treated with bmp- <dig> for various time points.
total protein lysates were prepared, and the amounts of the phosphorylated forms of smad1/5/ <dig> were determined by western blot analysis.
interestingly, treatment with  <dig> ng/ml bmp- <dig> induced phosphorylation of smad.
the bmp- <dig> induced phosphorylation was high at the earliest time point tested , and remained high for at least  <dig> hours .
a similar phosphorylation was observed in ramos cells, but not in hl <dig> cells .
furthermore, we also tested whether other known downstream signalling pathways of bmp- <dig> could be triggered by bmp- <dig> in human b cells.
however, we did not observe any significant changes in the level of phospho-stat <dig> or phospho-p <dig> upon bmp- <dig> treatment of b cells .
bmp- <dig> induces upregulation of id1
next, we wanted to explore whether the bmp- <dig> induced phosphorylation of smad 1/5/ <dig> also could induce transcriptional changes of target genes.
in this regard, the inhibitors of dna binding proteins  are considered to be some of the major target genes for smad-signalling  <cit> .
thus, b cells were pre-incubated over night in x-vivo  <dig>  and then cultured in medium alone or in the presence of bmp- <dig> for various time points before preparation of total rna.
the amount of id1–id <dig> mrna was quantified by real-time rt-pcr.
interestingly, we observed a specific four-fold upregulation of id <dig> mrna in bmp-6-treated b cells .
the up-regulation of id <dig> mrna was characteristic of an early inducible gene, with maximal upregulation two hours after the addition of bmp- <dig> and returned to baseline after  <dig> hours.
in contrast, no significant changes were observed for id <dig> and id <dig> mrna, whereas id4-transcripts were not detectable .
western blot analysis revealed that the bmp-6-induced upregulation of id <dig> mrna also was correlated with upregulation of id <dig> protein as well.
the increase in id <dig> protein level was detectable after one hour and increased until  <dig> hours after bmp- <dig> addition, showing a 16-fold upregulation compared with t <dig>  .
in line with the mrna data, no consistent change in the amounts of id <dig> and id <dig> protein could be observed .
we were able to block the id <dig> specific band with a blocking peptide .
taken together, these data suggest that id <dig> could be a possible target gene for mediating the effects of bmp- <dig> in human b cells, whereas id <dig> and id <dig> not seem to be involved.
bmp- <dig> production in b cells
the fact that bmp- <dig> has been reported to act as an autocrine stimulator in chondrocytes  <cit>  and ovarium  <cit> , prompted us to investigate whether normal human b cells could produce bmp- <dig> upon stimulation.
ramos cells, which have been described to express bmp- <dig> mrna endogenously  <cit> , and the t cell line jurkat, served as positive and negative controls, respectively.
endogenous bmp- <dig> mrna levels in normal b cells were quantified by real-time rt-pcr after stimulation with anti-igm for different time points.
interestingly, the up-regulation of bmp- <dig> mrna was characteristic of an early-to intermediate inducible gene with maximal upregulation four hours after the addition of anti-igm.
the level of bmp- <dig> mrna was back to baseline after  <dig> hours upon stimulation .
furthermore, both fcs and human ab-serum induced significant upregulation of bmp- <dig> mrna .
interestingly, in a separate study we have found that normal human t cells do not express bmp- <dig> mrna after activation .
next, we wanted to detect bmp- <dig> protein in normal b-cells and tested various commercially available antibodies.
however, in our hands these anti-bmp- <dig> antibodies did only recognize the recombinant bmp- <dig> protein and not the native protein.
discussion
recent studies have demonstrated an important role for bmp superfamily members in hematopoietic stem cells, early thymocytes  <cit>  and b-cell malignancies  <cit> , but a role for bmps in normal human b cells has previously not been reported.
the present study demonstrated a significant antiproliferative effect of bmp- <dig> in peripheral blood cd19+ b cells.
additionally, bmp- <dig> induced cell death in cd27+ memory b cells as well as in a burkitt lymphoma cell line .
importantly, bmp- <dig> induced a rapid and marked increase in smad-1/5/ <dig> phosphorylation.
furthermore, the bmp- <dig> induced smad phosphorylation was followed by a selective upregulation of id <dig> mrna and subsequent id <dig> protein.
in the present study, the demonstrated antiproliferative effect of bmp- <dig> in anti-igm treated b cells was significant and dose-dependent.
importantly, the anti-proliferative effect of bmp- <dig> could be completely neutralized by the use of a natural inhibitor, noggin.
this is in line with others, showing that noggin can function as a bmp- <dig> antagonist  <cit> .
in addition, the combination of soluble bmp-rib-fc and bmp-rii-fc fusion proteins also neutralized the anti-proliferative effect of bmp- <dig> in human b cells.
interestingly, as for other tgf family members, bifunctional effects have also been demonstrated for bmps.
whereas several of the bmps have been shown to promote proliferation in various cell types including condrocytes  <cit> , liver  <cit>  and granulosa cells  <cit> , antiproliferative effects and induction of apoptosis has been reported for b and t lineage cells.
similar effects as demonstrated for bmp- <dig> on human b cells in the present study, were demonstrated for bmp- <dig>   <dig>   <dig> and - <dig> in human myeloma cells  <cit> .
other members of the bmp-family have also been reported to induce apoptosis, including in mouse b lineage cells  <cit> .
additionally, bmp- <dig> inhibits thymocyte proliferation  <cit> .
taken together, these data suggest that the role of bmps in the regulation of proliferation and apoptosis is highly cell type dependent.
to examine how bmp- <dig> exerts its functional effects in b cells, we analysed bmp receptor expression by western blot analysis.
human peripheral b cells were found to express the bmp type i receptors act-ria and bmp-rib, and the type ii-receptors bmp-rii and act-riib, which signal after binding of several bmps, including bmp- <dig>  <cit> .
to further explore bmp- <dig> induced signalling, activation of several pathways is possible.
the major signalling pathway known to date, is activation of r-smads  <cit> .
in that respect, bmps have been shown to exert antiproliferative effects in b lineage cells via phosphorylation of r-smad  <cit> .
furthermore, bmp- <dig> has been shown to induce activation of stat <dig> in myeloma cells  <cit> .
however, phosphorylation of r-smad was not investigated in that study.
bmp- <dig> has also been shown to induce phosphorylation of p <dig>  <cit> .
thus, phosphorylation of p <dig>  stat <dig> and smad1/5/ <dig> represent important bmp-signalling pathways that mediated the effects of bmps and even cross-talk between these pathways has been reported  <cit> .
in the present study, we were not able to detect bmp-6-induced changes in the phosphorylation status of stat <dig> or p <dig> in human peripheral b cells.
instead, a rapid and marked phosphorylation of smad1/5/ <dig> was revealed.
in a parallel study, we have found that other bmps also induced phosphorylation of smad1/5/ <dig> in peripheral b cells .
we are currently pursuing microarray studies to identify the signalling pathways and target genes that are differently regulated by the various bmps in human b cells.
upregulation of id <dig> via smad1/5/ <dig> phosphorylation is a known mechanism for bmp- <dig> signalling in other cell systems  <cit>  and regulation of id-proteins is thought to be an important mechanism for smad-signalling  <cit> .
in the present study, real-time rt-pcr experiments revealed a specific four-fold upregulation for id <dig> in bmp-6-treated b cells, while the amount of id2–id <dig> remained unchanged.
in agreement with this, western blot analysis demonstrated an upregulation of id <dig> protein, while the amount of id <dig> and id <dig> protein levels remained unchanged.
previously, id <dig> has been considered not to be expressed in later developmental stages than pro-b cells  <cit> , and its constitutive expression has been reported to impair mouse b cell development  <cit> .
therefore, our demonstration of the time-dependent upregulation of id <dig> mrna and protein in mature normal human b cells is of particular interest.
in that respect, it is noteworthy that tgf-β signalling in early and mature b cells induces both id <dig> and id <dig> expression  <cit> , but not id <dig> .
interestingly, these results show that various members of the tgf-β family regulate id proteins differently.
id <dig> and id <dig> are considered to be the id proteins mainly expressed in mature b cells  <cit> .
the present study also found id <dig> and id <dig> protein in b cells to be more highly expressed than id <dig> in resting b cells.
however, bmp- <dig> did not induce significant changes in the protein expression of id <dig> and id <dig>  it is believed that id proteins block differentiation and promote proliferation in various cell types  <cit> .
id proteins act as dominant-negative inhibitors of e-proteins and pax <dig> function by forming dimers with these proteins, making them unable to bind dna.
it has been proposed that the balance among e-proteins, pax <dig> and id proteins might have an important role in activated b cells  <cit> .
in that respect, e-proteins have been implicated in both the promotion and inhibition of cell survival and growth at different points in lymphocyte development  <cit> .
the antiproliferative and death inducing effect of bmp- <dig> in b cells with concomitant upregulation of id <dig> protein is therefore in line with the view that id proteins are required for the induction of growth arrest and apoptosis in b-lymphocyte progenitors by tgf-β  <cit> .
furthermore, id proteins are known as important parts of signalling pathways involved in development, cell cycle and tumorigenesis  <cit> .
it is well established that various members of the id family are overexpressed in a range of human tumours and generally, id <dig> appears to be the family member most widely overexpressed in a variety of human malignancies  <cit> , including multiple myeloma  <cit> .
additionally, our findings that bmp- <dig> activates intracellular signalling pathways in human b cells might be of potential pathophysiological significance in lymphoma and inflammation.
high bmp- <dig> mrna expression in dlbcl has been shown to correlate to unfavourable outcome  <cit> .
in this respect, it is of interest that targeted expression of id <dig> to b-lymphocytes resulted in aberrant b cell development, massive apoptosis, and subsequent development of b cell lymphomas  <cit> .
moreover, bmp- <dig> has been suggested to play a role in rheumatoid arthritis   <cit>  and elevated levels of id <dig> and id <dig> have been found in the synovia of ra-patients  <cit> .
altogether, these results point to an important role for id proteins in the regulation of normal b cell homeostasis and in diseases, where b cells are involved.
it will therefore be important to further elucidate the role of id- <dig> in human b cells by selective over expression or inhibition of id- <dig> gene expression.
given the role of bmp- <dig> in mature human b cells demonstrated here, identification of bmp- <dig> producing cells in vivo with possibility of interaction with naïve and memory b cells might contribute to the understanding of mature b cell biology.
high bmp- <dig> mrna expression in dlbcl has been detected by gene expression profiling  <cit> .
furthermore, production of bmp- <dig> transcripts in normal activated b cells was detected in the same study.
of note, an autocrine bmp- <dig> loop has been reported by others in chondrocytes and in the ovarium  <cit> .
therefore, we wanted to explore the possibility for an autocrine bmp- <dig> loop in human b cells.
we analysed the expression bmp- <dig> mrna in peripheral blood b cells by real-time pcr, and report here the upregulation of endogenous bmp- <dig> transcripts after stimulation with fcs, human ab-serum and, most importantly, anti-igm.
however, our attempts to study bmp- <dig> protein levels were unsuccessful due to problems with unspecific binding of the anti-bmp- <dig> antibodies tested, and lack of specific staining in control cells known to express bmp- <dig> mrna.
in contrast, the recombinant protein was readily detected.
in that respect, few investigators have detected bmp- <dig> protein in humans, especially in non-pathogenic tissue.
the possibility of bmp- <dig> production in human b-cells is in line with a recent work that reported the production of bmp- <dig> in mouse b cells, infiltrating the bone marrow of mice with inflammatory arthritis  <cit> .
in this study, a role for bmps in the inflammatoric process of arthritis was suggested.
the upregulation of the bmp-6-transcripts after igm-crosslinking is of pathophysiologic interest  <cit> .
a loss of tgf-β-responsiveness has been suggested to be a critical contribution to malignant transformation  <cit>  and similar oncogenic mechanisms have been postulated for bmps.
lines of evidence suggest  <cit>  that at early stages of carcinogenesis, bmp- <dig> is not a tumour promoter, but suppresses benign and malignant tumour outgrowth.
these findings are in good agreement with previous findings for other tgf-β family members, including tgf-β <dig> and bmp- <dig>  <cit> , indicating that cellular context of the bmp target cell might define the various observed effects.
in contrast to the upregulation of bmp- <dig> transcript in b cells, we were not able to detect bmp- <dig> transcripts in human peripheral blood cd4+ or cd8+ t cells , consistent with the findings in t cell lines  <cit>  and t cells in mice  <cit> .
other potential bmp- <dig> sources for mature b cells in vivo might be other cells of the immune system or tissue with contact to the hematopoietic system.
one well recognized source for bmp- <dig> production is the human bone and bone marrow stroma  <cit> .
furthermore, it is noteworthy that human umbilical vein endothelial cells  highly express bmp- <dig> mrna  <cit> , and vascular endothelium has been reported to produce bmp- <dig>  <cit> .
these studies might imply a role for bmp- <dig> in transendothelial migration of b cells.
bmp- <dig> mrna has been demonstrated in murine macrophage cell lines, but not in humans  <cit> .
in accordance with these findings, other human cell lines of neutrophil and monocytic origin have been described to be negative for the bmp- <dig> transcript  <cit> .
to our knowledge, there is currently no report about bmp- <dig> production of human dendritic cells.
CONCLUSIONS
in conclusion, our results show that bmp- <dig> induces activation of intracellular smad signalling in mature human b-cells with consecutive production of id <dig> protein.
furthermore, we report that bmp- <dig> has an antiproliferative effect in b cells stimulated with anti-igm alone or the combined action of anti-igm and cd40l.
additionally, bmp- <dig> induces cell death in activated memory b cells and ramos cells.
taken together, these results provide a rationale to further examine the role of bmp- <dig> signalling in normal b cell biology as well as in pathologic conditions like b cell malignancies and autoimmune disorders.

 members of the transforming growth factor β superfamily play central roles in controlling cellular proliferation, differentiation, migration and apoptosis. these cytokines can be divided into three subgroups: tgf-β, the activins/inhibins, and the bone morphogenetic proteins, of which the latter constitute the largest family. bmps are 30– kda hetero- or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation. several studies have demonstrated an essential role of these proteins during embryogenesis, and more recently, also in adult tissues. tgf-β has been intensively studied in normal and malignant haematopoietic cells and is one of the most potent endogenous negative regulators known to date.. in contrast, the effect of bmps in the immune system has not been widely investigated. in that respect, bmp- - and - have been found to control differentiation of hematopoietic stem cells and early t cell development. bmp- has been reported to reduce the number of cobblestone-area-forming cells of normal human haematopoietic cells. furthermore, bmp- - and - had an antiproliferative and a proapoptotic effect on multiple myeloma cells. in addition, by gene expression profiling, bmp- significantly increased the predictive value for a multi-gene signature test and was associated with a poor outcome in diffuse large b cell lymphomas. bmp- like the other bmp members, signals through ligation and heterodimerzation of bmp type i and type ii serine-threonine kinase receptors, which subsequently propagates the signal downstream by phosphorylating smad proteins. bmp- can signal through the ligation of the type i receptors act-ria, bmp-ria, and bmp-rib and the type ii receptors bmp-rii, act-riia and act-riib, which lead to the phosphorylation of the receptor smads. the r- smads then form complexes with the co-smad and are translocated into the nucleus where they exert gene regulation. given the reported role of bmp- in b-cell malignancies and haematopoietic progenitor cells, we wanted to explore its potential role in normal human b cells. we studied the effects of bmp- on proliferation and apoptosis on resting and stimulated b cells. furthermore, the expression of bmp receptors and bmp- induced activation of the smad signalling pathway with subsequent regulation of the target genes id1–id were resolved. finally, we investigated whether b cells also were capable of producing bmp-  bmp- inhibits anti-igm induced proliferation of human b cells the effects of bmp- on normal and neoplastic hematopoietic cells prompted us to investigate the effects of bmp- on normal human b cells. all experiments in this study were performed under serum-free conditions as fcs has been shown to interfere with bmp-signalling. to study the effect of bmp- on proliferation, b-cells from healthy volunteers were stimulated with anti-igm and/or cd40l in the presence or absence of bmp- for three days. we found that bmp- led to a 35% mean reduction of anti-igm- induced dna synthesis. similar results were obtained for b cells treated with anti-igm and cd40l. the bmp-6-induced inhibition of proliferation was dose-dependent in both peripheral b cells and the burkitt lymphoma cell line ramos. the bmp- effects could be reversed by addition of the extracellular inhibitor noggin. similarly, a combination of the soluble bmp receptors bmp-rib-fc and bmp-rii-fc also neutralized the effects of bmp-. next, we wanted to test whether bmp- had different effect on naïve and memory b cells. naïve and memory b cells were isolated from peripheral blood by cell sorting of immunobead-isolated cd19+ b cells, and tested for their capacity to proliferate in the presence of bmp- however, bmp- inhibited anti-igm induced dna synthesis in the two subpopulations to a similar extent, with a mean reduction of dna-synthesis of 45% for naïve b cells and 48% for memory b cells. bmp- induces cell death in human memory b cells and ramos cells next, we wanted to establish whether bmp- also could affect the viability of normal b cells. cell viability was determined by propidium iodide staining after culture with or without bmp- for hours. interestingly, bmp- showed a small, but reproducible mean increase of cell death from to 23% in anti-igm stimulated cd27+ memory b cells. furthermore, ramos cells showed a mean increase in cell death from to 50% after bmp- treatment. in contrast, cell death of total cd19+ cells or cd27-igg- naïve b cells was not significantly affected. human b cells express bmp- receptors detailed knowledge regarding expression of different bmp receptors in b cells is currently not available. to further elucidate the role of bmps in human b cells, we performed western blot analysis for type i and type ii bmp receptors. this analysis revealed that the type i receptors act-ria, bmp-rib and the type ii receptors bmp-rii and act-riib are expressed on resting human b-cells. ramos cells expressed the type i receptors act-ria, weakly bmp-rib and the type ii receptor bmp-rii, but more weakly than normal b cells. hl cells were used for comparison and weakly expressed act-ria and bmp-rii. taken together, these data show that normal human b cells and ramos cells express a set of bmp receptors, previously shown to bind bmp-. bmp- induces phosphorylation of smad1/5/8 upon ligand binding, the type ii receptor transphosphorylates and activates the type i receptor. type i receptors can signal via several pathways. we examined the effect of bmp- on smad phosphorylation, as the activation of smad is considered to be a major signalling pathway for bmps. b cells were cultured in serum-free media over night and then treated with bmp- for various time points. total protein lysates were prepared, and the amounts of the phosphorylated forms of smad1/5/ were determined by western blot analysis. interestingly, treatment with ng/ml bmp- induced phosphorylation of smad. the bmp- induced phosphorylation was high at the earliest time point tested, and remained high for at least hours. a similar phosphorylation was observed in ramos cells, but not in hl cells. furthermore, we also tested whether other known downstream signalling pathways of bmp- could be triggered by bmp- in human b cells. however, we did not observe any significant changes in the level of phospho-stat or phospho-p upon bmp- treatment of b cells. bmp- induces upregulation of id1 next, we wanted to explore whether the bmp- induced phosphorylation of smad 1/5/ also could induce transcriptional changes of target genes. in this regard, the inhibitors of dna binding proteins are considered to be some of the major target genes for smad-signalling. thus, b cells were pre-incubated over night in x-vivo and then cultured in medium alone or in the presence of bmp- for various time points before preparation of total rna. the amount of id1–id mrna was quantified by real-time rt-pcr. interestingly, we observed a specific four-fold upregulation of id mrna in bmp-6-treated b cells. the up-regulation of id mrna was characteristic of an early inducible gene, with maximal upregulation two hours after the addition of bmp- and returned to baseline after hours. in contrast, no significant changes were observed for id and id mrna, whereas id4-transcripts were not detectable. western blot analysis revealed that the bmp-6-induced upregulation of id mrna also was correlated with upregulation of id protein as well. the increase in id protein level was detectable after one hour and increased until hours after bmp- addition, showing a 16-fold upregulation compared with t. in line with the mrna data, no consistent change in the amounts of id and id protein could be observed. we were able to block the id specific band with a blocking peptide. taken together, these data suggest that id could be a possible target gene for mediating the effects of bmp- in human b cells, whereas id and id not seem to be involved. bmp- production in b cells the fact that bmp- has been reported to act as an autocrine stimulator in chondrocytes and ovarium, prompted us to investigate whether normal human b cells could produce bmp- upon stimulation. ramos cells, which have been described to express bmp- mrna endogenously, and the t cell line jurkat, served as positive and negative controls, respectively. endogenous bmp- mrna levels in normal b cells were quantified by real-time rt-pcr after stimulation with anti-igm for different time points. interestingly, the up-regulation of bmp- mrna was characteristic of an early-to intermediate inducible gene with maximal upregulation four hours after the addition of anti-igm. the level of bmp- mrna was back to baseline after hours upon stimulation. furthermore, both fcs and human ab-serum induced significant upregulation of bmp- mrna. interestingly, in a separate study we have found that normal human t cells do not express bmp- mrna after activation. next, we wanted to detect bmp- protein in normal b-cells and tested various commercially available antibodies. however, in our hands these anti-bmp- antibodies did only recognize the recombinant bmp- protein and not the native protein. discussion recent studies have demonstrated an important role for bmp superfamily members in hematopoietic stem cells, early thymocytes and b-cell malignancies, but a role for bmps in normal human b cells has previously not been reported. the present study demonstrated a significant antiproliferative effect of bmp- in peripheral blood cd19+ b cells. additionally, bmp- induced cell death in cd27+ memory b cells as well as in a burkitt lymphoma cell line. importantly, bmp- induced a rapid and marked increase in smad-1/5/ phosphorylation. furthermore, the bmp- induced smad phosphorylation was followed by a selective upregulation of id mrna and subsequent id protein. in the present study, the demonstrated antiproliferative effect of bmp- in anti-igm treated b cells was significant and dose-dependent. importantly, the anti-proliferative effect of bmp- could be completely neutralized by the use of a natural inhibitor, noggin. this is in line with others, showing that noggin can function as a bmp- antagonist. in addition, the combination of soluble bmp-rib-fc and bmp-rii-fc fusion proteins also neutralized the anti-proliferative effect of bmp- in human b cells. interestingly, as for other tgf family members, bifunctional effects have also been demonstrated for bmps. whereas several of the bmps have been shown to promote proliferation in various cell types including condrocytes, liver and granulosa cells, antiproliferative effects and induction of apoptosis has been reported for b and t lineage cells. similar effects as demonstrated for bmp- on human b cells in the present study, were demonstrated for bmp- and - in human myeloma cells. other members of the bmp-family have also been reported to induce apoptosis, including in mouse b lineage cells. additionally, bmp- inhibits thymocyte proliferation. taken together, these data suggest that the role of bmps in the regulation of proliferation and apoptosis is highly cell type dependent. to examine how bmp- exerts its functional effects in b cells, we analysed bmp receptor expression by western blot analysis. human peripheral b cells were found to express the bmp type i receptors act-ria and bmp-rib, and the type ii-receptors bmp-rii and act-riib, which signal after binding of several bmps, including bmp-. to further explore bmp- induced signalling, activation of several pathways is possible. the major signalling pathway known to date, is activation of r-smads. in that respect, bmps have been shown to exert antiproliferative effects in b lineage cells via phosphorylation of r-smad. furthermore, bmp- has been shown to induce activation of stat in myeloma cells. however, phosphorylation of r-smad was not investigated in that study. bmp- has also been shown to induce phosphorylation of p. thus, phosphorylation of p stat and smad1/5/ represent important bmp-signalling pathways that mediated the effects of bmps and even cross-talk between these pathways has been reported. in the present study, we were not able to detect bmp-6-induced changes in the phosphorylation status of stat or p in human peripheral b cells. instead, a rapid and marked phosphorylation of smad1/5/ was revealed. in a parallel study, we have found that other bmps also induced phosphorylation of smad1/5/ in peripheral b cells. we are currently pursuing microarray studies to identify the signalling pathways and target genes that are differently regulated by the various bmps in human b cells. upregulation of id via smad1/5/ phosphorylation is a known mechanism for bmp- signalling in other cell systems and regulation of id-proteins is thought to be an important mechanism for smad-signalling. in the present study, real-time rt-pcr experiments revealed a specific four-fold upregulation for id in bmp-6-treated b cells, while the amount of id2–id remained unchanged. in agreement with this, western blot analysis demonstrated an upregulation of id protein, while the amount of id and id protein levels remained unchanged. previously, id has been considered not to be expressed in later developmental stages than pro-b cells, and its constitutive expression has been reported to impair mouse b cell development. therefore, our demonstration of the time-dependent upregulation of id mrna and protein in mature normal human b cells is of particular interest. in that respect, it is noteworthy that tgf-β signalling in early and mature b cells induces both id and id expression, but not id. interestingly, these results show that various members of the tgf-β family regulate id proteins differently. id and id are considered to be the id proteins mainly expressed in mature b cells. the present study also found id and id protein in b cells to be more highly expressed than id in resting b cells. however, bmp- did not induce significant changes in the protein expression of id and id it is believed that id proteins block differentiation and promote proliferation in various cell types. id proteins act as dominant-negative inhibitors of e-proteins and pax function by forming dimers with these proteins, making them unable to bind dna. it has been proposed that the balance among e-proteins, pax and id proteins might have an important role in activated b cells. in that respect, e-proteins have been implicated in both the promotion and inhibition of cell survival and growth at different points in lymphocyte development. the antiproliferative and death inducing effect of bmp- in b cells with concomitant upregulation of id protein is therefore in line with the view that id proteins are required for the induction of growth arrest and apoptosis in b-lymphocyte progenitors by tgf-β. furthermore, id proteins are known as important parts of signalling pathways involved in development, cell cycle and tumorigenesis. it is well established that various members of the id family are overexpressed in a range of human tumours and generally, id appears to be the family member most widely overexpressed in a variety of human malignancies, including multiple myeloma. additionally, our findings that bmp- activates intracellular signalling pathways in human b cells might be of potential pathophysiological significance in lymphoma and inflammation. high bmp- mrna expression in dlbcl has been shown to correlate to unfavourable outcome. in this respect, it is of interest that targeted expression of id to b-lymphocytes resulted in aberrant b cell development, massive apoptosis, and subsequent development of b cell lymphomas. moreover, bmp- has been suggested to play a role in rheumatoid arthritis and elevated levels of id and id have been found in the synovia of ra-patients. altogether, these results point to an important role for id proteins in the regulation of normal b cell homeostasis and in diseases, where b cells are involved. it will therefore be important to further elucidate the role of id- in human b cells by selective over expression or inhibition of id- gene expression. given the role of bmp- in mature human b cells demonstrated here, identification of bmp- producing cells in vivo with possibility of interaction with naïve and memory b cells might contribute to the understanding of mature b cell biology. high bmp- mrna expression in dlbcl has been detected by gene expression profiling. furthermore, production of bmp- transcripts in normal activated b cells was detected in the same study. of note, an autocrine bmp- loop has been reported by others in chondrocytes and in the ovarium. therefore, we wanted to explore the possibility for an autocrine bmp- loop in human b cells. we analysed the expression bmp- mrna in peripheral blood b cells by real-time pcr, and report here the upregulation of endogenous bmp- transcripts after stimulation with fcs, human ab-serum and, most importantly, anti-igm. however, our attempts to study bmp- protein levels were unsuccessful due to problems with unspecific binding of the anti-bmp- antibodies tested, and lack of specific staining in control cells known to express bmp- mrna. in contrast, the recombinant protein was readily detected. in that respect, few investigators have detected bmp- protein in humans, especially in non-pathogenic tissue. the possibility of bmp- production in human b-cells is in line with a recent work that reported the production of bmp- in mouse b cells, infiltrating the bone marrow of mice with inflammatory arthritis. in this study, a role for bmps in the inflammatoric process of arthritis was suggested. the upregulation of the bmp-6-transcripts after igm-crosslinking is of pathophysiologic interest. a loss of tgf-β-responsiveness has been suggested to be a critical contribution to malignant transformation and similar oncogenic mechanisms have been postulated for bmps. lines of evidence suggest that at early stages of carcinogenesis, bmp- is not a tumour promoter, but suppresses benign and malignant tumour outgrowth. these findings are in good agreement with previous findings for other tgf-β family members, including tgf-β and bmp-, indicating that cellular context of the bmp target cell might define the various observed effects. in contrast to the upregulation of bmp- transcript in b cells, we were not able to detect bmp- transcripts in human peripheral blood cd4+ or cd8+ t cells, consistent with the findings in t cell lines and t cells in mice. other potential bmp- sources for mature b cells in vivo might be other cells of the immune system or tissue with contact to the hematopoietic system. one well recognized source for bmp- production is the human bone and bone marrow stroma. furthermore, it is noteworthy that human umbilical vein endothelial cells highly express bmp- mrna, and vascular endothelium has been reported to produce bmp-. these studies might imply a role for bmp- in transendothelial migration of b cells. bmp- mrna has been demonstrated in murine macrophage cell lines, but not in humans. in accordance with these findings, other human cell lines of neutrophil and monocytic origin have been described to be negative for the bmp- transcript. to our knowledge, there is currently no report about bmp- production of human dendritic cells.  in conclusion, our results show that bmp- induces activation of intracellular smad signalling in mature human b-cells with consecutive production of id protein. furthermore, we report that bmp- has an antiproliferative effect in b cells stimulated with anti-igm alone or the combined action of anti-igm and cd40l. additionally, bmp- induces cell death in activated memory b cells and ramos cells. taken together, these results provide a rationale to further examine the role of bmp- signalling in normal b cell biology as well as in pathologic conditions like b cell malignancies and autoimmune disorders. 
a potential role of bmp- in the immune system has been implied by various studies of malignant and rheumatoid diseases. furthermore, we found that bmp- had an antiproliferative effect both in naïve and memory b cells stimulated with anti-igm alone or the combined action of anti-igm and cd40l. additionally, bmp- induced cell death in activated memory b cells. in the present study, we explored the role of bmp- in normal human peripheral blood b cells. the b cells were found to express bmp type i and type ii receptors and bmp- rapidly induced phosphorylation of smad1/5/ furthermore, smad-phosphorylation was followed by upregulation of id mrna and id protein, whereas id and id expression was not affected. furthermore, b cells were demonstrated to upregulate bmp- mrna upon stimulation with anti-igm. in mature human b cells, bmp- inhibited cell growth, and rapidly induced phosphorylation of smad1/5/ followed by an upregulation of id importantly, the antiproliferative effect of bmp- in b-cells was completely neutralized by the natural antagonist, noggin. bone morphogenetic proteins belong to the tgf-β superfamily and are secreted proteins with pleiotropic roles in many different cell types.
 bone morphogenetic proteins belong to the tgf-β superfamily and are secreted proteins with pleiotropic roles in many different cell types. a potential role of bmp- in the immune system has been implied by various studies of malignant and rheumatoid diseases. in the present study, we explored the role of bmp- in normal human peripheral blood b cells.  the b cells were found to express bmp type i and type ii receptors and bmp- rapidly induced phosphorylation of smad1/5/ furthermore, smad-phosphorylation was followed by upregulation of id mrna and id protein, whereas id and id expression was not affected. furthermore, we found that bmp- had an antiproliferative effect both in naïve and memory b cells stimulated with anti-igm alone or the combined action of anti-igm and cd40l. additionally, bmp- induced cell death in activated memory b cells. importantly, the antiproliferative effect of bmp- in b-cells was completely neutralized by the natural antagonist, noggin. furthermore, b cells were demonstrated to upregulate bmp- mrna upon stimulation with anti-igm.  in mature human b cells, bmp- inhibited cell growth, and rapidly induced phosphorylation of smad1/5/ followed by an upregulation of id 