BACKGROUND
cell migration and chemotaxis that occur in malignancies and inflammatory processes may deposit the focal adhesion component α-actinin in their migratory path  <cit> .
we previously showed that extracellular α-actinin is degraded by monocyte-secreted urokinase to generate a specific fragment   <cit> .
mactinin is found at various sites of monocytic activation in vivo  <cit> , has chemotactic activity for monocytes  <cit>  and promotes monocyte/macrophage maturation  <cit> .
these findings suggest that mactinin is a functionally important mediator of monocytic activity.
monocytes and macrophages play pivotal roles during inflammatory and immune processes by releasing various cytokines including tumor necrosis factor -α, interleukin -1α and il-1β, chemokines, enzymes and other factors  <cit> .
in some disease processes such as infections  <cit>  and wound healing  <cit> , macrophage activity may be beneficial in promoting healing.
in other diseases, such as arthritis  <cit>  and atherosclerosis  <cit> , macrophage activation may contribute to pathogenesis and propagation.
the monocyte/macrophage system also plays an integral role in malignancies by secretion of these cytokines, generation of dendritic cells and osteoclasts and modulation of the immune response .
in the current study, we examined the mechanism mediating the stimulatory effect of mactinin on monocytes.
we show here that mactinin binds to a heterocomplex including heat shock protein - <dig> on monocytes, and that hsp <dig> is critically important for the stimulatory activity of mactinin on monocytes since inhibition of hsp <dig> almost completely blocked mactinin-induced cytokine production and migration of monocytes.
hsp <dig> is a molecular chaperone whose activity promotes chemotaxis, migration, proliferation and cytokine secretion in malignant and endothelial cells and in monocytes  <cit> .
our identification of mactinin as a novel inducer of hsp <dig> activity on monocytes therefore has important implications for diverse conditions including malignancies, autoimmune disease, inflammation and atherosclerosis.
RESULTS
mactinin stimulates il-1α, il-1β and tnf-α production by monocytes
peripheral blood monocytes were isolated and cultured for  <dig> h with  <dig> nm mactinin,  <dig> nm α-actinin,  <dig> nm gst or medium alone .
the gst condition was included in order to control for the 10% contaminating gst in our mactinin preparation.
supernatants were recovered and centrifuged to remove nonadherent cells and aliquots assayed for the  <dig> cytokines.
as shown in fig.
<dig>  the levels of il-1α, il-1β, and tnfα were significantly increased in the supernatants of mactinin-treated monocytes.
control cultures treated with α-actinin or gst did not show any increase in cytokine production.
mactinin did not stimulate the production of granulocyte macrophage colony-stimulating factor , interferon -α, il- <dig>  macrophage colony-stimulating factor , or macrophage inhibitory protein -1α .
these findings indicate that mactinin directly stimulates the production of specific pro-inflammatory cytokines from monocytes.
mactinin binds to monocytes
to assess whether mactinin binds to peripheral blood monocytes, these cells were incubated with or without mactinin and then stained with antiserum to mactinin or isotype matched  control pre-immune antiserum.
bound mactinin was measured using flow cytometry .
there was a significantly higher percentage of positively staining monocytes that had been incubated with mactinin  than monocytes not incubated with mactinin  or monocytes incubated with mactinin but detected with control igg <dig> antiserum .
furthermore, the mean fluorescence intensity of monocytes incubated with mactinin  was significantly higher than that of monocytes incubated without mactinin  or monocytes incubated with mactinin but detected with control igg <dig> antiserum .
we saw a similar increase in positive staining and mean fluorescence in hl- <dig> cells incubated with mactinin .
thus, mactinin binds to both monocytes and monocytoid cells.
mactinin binds to a specific high molecular weight complex on hl- <dig> cells
we next examined the specificity of mactinin binding to hl- <dig> cells, by determining the ability of excess unlabeled  mactinin to compete for the binding of mactinin to hl- <dig> cells.
hl- <dig> cells were pre-incubated with or without unlabeled mactinin, followed by addition of mactinin.
autoradiography of the clarified lysates resolved by 12% non-reducing sds-page detected a high molecular weight complex on cells that were incubated with mactinin .
this complex was markedly reduced in cells pre-incubated with unlabeled mactinin .
these results suggest that mactinin can compete for a specific and potentially saturable binding site on hl- <dig> cells.
mactinin binds a hetero-complex of hl- <dig> cell membrane proteins including hsp90
mactinin has in vitro maturation promoting effects on both monocytes and hl- <dig> leukemia cells  <cit> .
we therefore elected to use the plentiful hl- <dig> cell line for biochemical analysis of mactinin binding proteins.
biotin surface labeled hl- <dig> cells were incubated with  <dig> nm mactinin in the presence or absence of the cleavable, cell-permeable cross-linker dsp or the non-cleavable, cell-impermeable cross-linker bs <dig>  the cells were lysed, centrifuged and proteins in the supernatant resolved by 6–20% gradient sds-page under reducing or non-reducing conditions.
autoradiography detected the  <dig> kd mactinin that was bound to hl- <dig> cells in the absence of a cross-linker .
use of the cross-linker dsp demonstrated that most of the mactinin bound to a high molecular weight complex on hl- <dig> cells .
under reducing conditions, the free mactinin could be separated out completely from the hl- <dig> protein complex seen with dsp .
since bs <dig> is not cleavable, the complex remained unchanged  under reducing conditions with bs <dig> .
to identify hl- <dig> proteins in the binding complex, the radiolabeled complex  was cut, electroeluted, dialyzed, reduced with dtt, run on a column of mactinin covalently bound to an affi-gel  <dig> matrix and eluted to select for mactinin-binding proteins.
a purification step was necessary since mass spectrometry analysis of the resulting proteins from the unpurified complex showed many co-migrating proteins including myeloperoxidase, glucose-regulated protein  <dig>   <dig> kd glucose-regulated protein, calnexin, calreticulin precursor, moesin, hsc70-ps <dig>  protein disulfide isomerase-associated  <dig>  prolyl 4-hydroxylase beta subunit and protein disulfide isomerase-associated  <dig> precursor.
nevertheless, even in the unpurified complex, more peptides matched hsp <dig>  than any other single protein.
the biotin-labeled hl- <dig> proteins that were eluted from matrix-bound mactinin were separated by sds-page and transferred to a pvdf membrane for detection of the proteins by western immunoblotting for biotin.
as shown in fig.
2d, three protein bands of molecular weight  <dig>   <dig> and  <dig> kd were detected.
a companion gel lane stained with coomassie-blue visualized only the  <dig> kd band .
the identity of the  <dig> kd protein was confirmed by western immunoblotting.
re-probing the membrane shown in fig.
2d with antiserum to hsp <dig> detected a single band of  <dig> kd , as well as recombinant hsp <dig> run in a separate lane as a positive control .
together, the above data indicate that mactinin binds to a high molecular weight complex of at least  <dig> proteins on monocytoid hl- <dig> cells, and one of these proteins is hsp <dig>
hsp <dig> mediates the stimulation of monocytes by mactinin
to determine if hsp <dig> identified in the mactinin-binding complex is functionally involved in the activity of mactinin on monocytes, we assessed the effect of  <dig> separate hsp <dig> inhibitors on mactinin-induced monocyte migration  <cit>  and cytokine production.
as shown in fig.
<dig>  all three hsp <dig> inhibitors -17-demethoxygeldanamycin , completely suppressed monocyte chemotaxis, since the number of cells that migrated towards mactinin in the presence of the hsp <dig> inhibitors was comparable to the number of cells that migrated in medium alone .
in contrast, fmlp-induced chemotaxis was not inhibited.
the concentrations of the hsp <dig> inhibitors tested included  <dig>   <dig>  and  <dig> nm geldanamycin,  <dig>  and  <dig> μm 17-aag, and  <dig> nm 17-dmag, based on previous reports of in vitro hsp <dig> inhibition studies  <cit> .
all concentrations significantly inhibited migration without meaningful differences .
finally, we examined if the same hsp <dig> inhibitors block mactinin-induced inflammatory cytokine production from monocytes .
each of the three hsp <dig> inhibitors completely blocked the production of il-1α and il-1β from monocytes.
in contrast, production of tnf-α was not completely blocked since tnf-α levels in the presence of the hsp <dig> inhibitors were higher than in medium alone, though it was markedly reduced compared to the levels seen with mactinin stimulation.
discussion
this study shows that mactinin induces hsp90-mediated monocyte activation resulting in stimulation of cytokine secretion and migration.
the recognition that degradative products of cytoskeletal proteins may influence hsp <dig> activity in target cells has important implications for diverse conditions including malignancies, autoimmune disease, inflammation and atherosclerosis.
our findings also extend the current understanding of "matrikines," proteolytically released extracellular matrix-derived bioactive peptides that influence many types of cells and processes .
hsp <dig> is a highly conserved member of the group of molecular chaperones, is usually cytosolic  <cit>  but may also be associated with lipid rafts in the cell membrane  <cit> .
hsps are constitutively expressed but enhanced in response to cellular stresses  <cit> .
their major function appears to be to act as protein chaperones, facilitating folding, oligomeric assembly, transport to a particular subcellular compartment, or switching between active and inactive protein conformation  <cit> .
hsp <dig> regulates many client proteins involved in signal transduction  <cit> .
other important functions include stimulation of chemotaxis, migration, proliferation and cytokine secretion in malignant and endothelial cells  <cit> .
we show for the first time that hsp <dig> is a functionally critical component of the mactinin-binding heterocomplex on monocytic cells.
two other proteins of molecular weight  <dig> and  <dig> kd found in this complex may possibly represent other hsps, since previous studies have shown that hsp90/ <dig> forms a receptor complex in monocytes and macrophages  <cit> .
many hsps interact with components of the actin cytoskeleton, perhaps modulating the cytoskeletal response to stress  <cit> .
it has been reported that hsp <dig> and hsp <dig> bind actin  <cit> , while the smaller hsp <dig> has been reported to associate with both actin and α-actinin  <cit> .
however, it is likely that the direct binding of extracellular mactinin is to other  protein on the surface of hl- <dig> cells, which then form a hetero-complex with hsp <dig>
in monocytes/macrophages and related cells, hsp <dig> potently induces tnf-α and il-1β expression and activation  <cit>  and cell cycling and differentiation  <cit> .
the hsp-activated macrophage axis may also play an important role in atherosclerosis .
inhibitors of hsp <dig>  such as geldanamycin and its analogues, have been used to identify hsp <dig> substrates  <cit> .
we found that geldanamycin and two of its analogues markedly inhibited mactinin-induced chemotaxis and cytokine production.
their ability to inhibit chemotaxis was specific for mactinin since they had no significant effect on fmlp-induced chemotaxis.
we also found that hsp <dig> inhibitors blocked mactinin-induced cytokine production from monocytes, confirming that hsp <dig> mediates the stimulatory activity of mactinin on monocytes.
our results suggest that a cytoskeletal protein derivative  may propagate inflammation, in part, by stimulating production of the pro-inflammatory cytokines il-1α, il-1β and tnf-α.
these two cytokines are elevated in the serum and synovial fluid of patients with rheumatoid arthritis, and both are capable of inducing joint damage in experimental arthritis models  <cit> .
the clinical efficacy of agents that block these cytokines in ameliorating bone and joint destruction in arthritis is now widely accepted  <cit> .
hsp <dig> induces urokinase-type plasminogen activator secretion  <cit>  and urokinase secreted by activated monocytes degrades α-actinin to generate mactinin  <cit> .
it is therefore possible that the binding and stimulation of hsp <dig> activity on monocytes by mactinin itself  may represent a positive feed-forward loop that further amplifies the immune/inflammatory response.
we therefore speculate that agents that block mactinin may provide a novel strategy for inhibiting hsp <dig> activity in diverse diseases.
CONCLUSIONS
we have identified that hsp <dig> mediates the stimulation of monocytes by mactinin, a fragment of the cytoskeletal protein α-actinin.
mactinin represents a novel inducer of hsp <dig> activity on monocytes.
our findings suggest that inhibition of mactinin may be of benefit in diseases propagated by monocyte/macrophage activation or hsp <dig> activity.

 cell migration and chemotaxis that occur in malignancies and inflammatory processes may deposit the focal adhesion component α-actinin in their migratory path. we previously showed that extracellular α-actinin is degraded by monocyte-secreted urokinase to generate a specific fragment. mactinin is found at various sites of monocytic activation in vivo, has chemotactic activity for monocytes and promotes monocyte/macrophage maturation. these findings suggest that mactinin is a functionally important mediator of monocytic activity. monocytes and macrophages play pivotal roles during inflammatory and immune processes by releasing various cytokines including tumor necrosis factor -α, interleukin -1α and il-1β, chemokines, enzymes and other factors. in some disease processes such as infections and wound healing, macrophage activity may be beneficial in promoting healing. in other diseases, such as arthritis and atherosclerosis, macrophage activation may contribute to pathogenesis and propagation. the monocyte/macrophage system also plays an integral role in malignancies by secretion of these cytokines, generation of dendritic cells and osteoclasts and modulation of the immune response. in the current study, we examined the mechanism mediating the stimulatory effect of mactinin on monocytes. we show here that mactinin binds to a heterocomplex including heat shock protein - on monocytes, and that hsp is critically important for the stimulatory activity of mactinin on monocytes since inhibition of hsp almost completely blocked mactinin-induced cytokine production and migration of monocytes. hsp is a molecular chaperone whose activity promotes chemotaxis, migration, proliferation and cytokine secretion in malignant and endothelial cells and in monocytes. our identification of mactinin as a novel inducer of hsp activity on monocytes therefore has important implications for diverse conditions including malignancies, autoimmune disease, inflammation and atherosclerosis.  mactinin stimulates il-1α, il-1β and tnf-α production by monocytes peripheral blood monocytes were isolated and cultured for h with nm mactinin, nm α-actinin, nm gst or medium alone. the gst condition was included in order to control for the 10% contaminating gst in our mactinin preparation. supernatants were recovered and centrifuged to remove nonadherent cells and aliquots assayed for the cytokines. as shown in fig. the levels of il-1α, il-1β, and tnfα were significantly increased in the supernatants of mactinin-treated monocytes. control cultures treated with α-actinin or gst did not show any increase in cytokine production. mactinin did not stimulate the production of granulocyte macrophage colony-stimulating factor, interferon -α, il- macrophage colony-stimulating factor, or macrophage inhibitory protein -1α. these findings indicate that mactinin directly stimulates the production of specific pro-inflammatory cytokines from monocytes. mactinin binds to monocytes to assess whether mactinin binds to peripheral blood monocytes, these cells were incubated with or without mactinin and then stained with antiserum to mactinin or isotype matched control pre-immune antiserum. bound mactinin was measured using flow cytometry. there was a significantly higher percentage of positively staining monocytes that had been incubated with mactinin than monocytes not incubated with mactinin or monocytes incubated with mactinin but detected with control igg antiserum. furthermore, the mean fluorescence intensity of monocytes incubated with mactinin was significantly higher than that of monocytes incubated without mactinin or monocytes incubated with mactinin but detected with control igg antiserum. we saw a similar increase in positive staining and mean fluorescence in hl- cells incubated with mactinin. thus, mactinin binds to both monocytes and monocytoid cells. mactinin binds to a specific high molecular weight complex on hl- cells we next examined the specificity of mactinin binding to hl- cells, by determining the ability of excess unlabeled mactinin to compete for the binding of mactinin to hl- cells. hl- cells were pre-incubated with or without unlabeled mactinin, followed by addition of mactinin. autoradiography of the clarified lysates resolved by 12% non-reducing sds-page detected a high molecular weight complex on cells that were incubated with mactinin. this complex was markedly reduced in cells pre-incubated with unlabeled mactinin. these results suggest that mactinin can compete for a specific and potentially saturable binding site on hl- cells. mactinin binds a hetero-complex of hl- cell membrane proteins including hsp90 mactinin has in vitro maturation promoting effects on both monocytes and hl- leukemia cells. we therefore elected to use the plentiful hl- cell line for biochemical analysis of mactinin binding proteins. biotin surface labeled hl- cells were incubated with nm mactinin in the presence or absence of the cleavable, cell-permeable cross-linker dsp or the non-cleavable, cell-impermeable cross-linker bs the cells were lysed, centrifuged and proteins in the supernatant resolved by 6–20% gradient sds-page under reducing or non-reducing conditions. autoradiography detected the kd mactinin that was bound to hl- cells in the absence of a cross-linker. use of the cross-linker dsp demonstrated that most of the mactinin bound to a high molecular weight complex on hl- cells. under reducing conditions, the free mactinin could be separated out completely from the hl- protein complex seen with dsp. since bs is not cleavable, the complex remained unchanged under reducing conditions with bs. to identify hl- proteins in the binding complex, the radiolabeled complex was cut, electroeluted, dialyzed, reduced with dtt, run on a column of mactinin covalently bound to an affi-gel matrix and eluted to select for mactinin-binding proteins. a purification step was necessary since mass spectrometry analysis of the resulting proteins from the unpurified complex showed many co-migrating proteins including myeloperoxidase, glucose-regulated protein kd glucose-regulated protein, calnexin, calreticulin precursor, moesin, hsc70-ps protein disulfide isomerase-associated prolyl 4-hydroxylase beta subunit and protein disulfide isomerase-associated precursor. nevertheless, even in the unpurified complex, more peptides matched hsp than any other single protein. the biotin-labeled hl- proteins that were eluted from matrix-bound mactinin were separated by sds-page and transferred to a pvdf membrane for detection of the proteins by western immunoblotting for biotin. as shown in fig. 2d, three protein bands of molecular weight and kd were detected. a companion gel lane stained with coomassie-blue visualized only the kd band. the identity of the kd protein was confirmed by western immunoblotting. re-probing the membrane shown in fig. 2d with antiserum to hsp detected a single band of kd, as well as recombinant hsp run in a separate lane as a positive control. together, the above data indicate that mactinin binds to a high molecular weight complex of at least proteins on monocytoid hl- cells, and one of these proteins is hsp hsp mediates the stimulation of monocytes by mactinin to determine if hsp identified in the mactinin-binding complex is functionally involved in the activity of mactinin on monocytes, we assessed the effect of separate hsp inhibitors on mactinin-induced monocyte migration and cytokine production. as shown in fig. all three hsp inhibitors -17-demethoxygeldanamycin, completely suppressed monocyte chemotaxis, since the number of cells that migrated towards mactinin in the presence of the hsp inhibitors was comparable to the number of cells that migrated in medium alone. in contrast, fmlp-induced chemotaxis was not inhibited. the concentrations of the hsp inhibitors tested included and nm geldanamycin, and μm 17-aag, and nm 17-dmag, based on previous reports of in vitro hsp inhibition studies. all concentrations significantly inhibited migration without meaningful differences. finally, we examined if the same hsp inhibitors block mactinin-induced inflammatory cytokine production from monocytes. each of the three hsp inhibitors completely blocked the production of il-1α and il-1β from monocytes. in contrast, production of tnf-α was not completely blocked since tnf-α levels in the presence of the hsp inhibitors were higher than in medium alone, though it was markedly reduced compared to the levels seen with mactinin stimulation. discussion this study shows that mactinin induces hsp90-mediated monocyte activation resulting in stimulation of cytokine secretion and migration. the recognition that degradative products of cytoskeletal proteins may influence hsp activity in target cells has important implications for diverse conditions including malignancies, autoimmune disease, inflammation and atherosclerosis. our findings also extend the current understanding of "matrikines," proteolytically released extracellular matrix-derived bioactive peptides that influence many types of cells and processes. hsp is a highly conserved member of the group of molecular chaperones, is usually cytosolic but may also be associated with lipid rafts in the cell membrane. hsps are constitutively expressed but enhanced in response to cellular stresses. their major function appears to be to act as protein chaperones, facilitating folding, oligomeric assembly, transport to a particular subcellular compartment, or switching between active and inactive protein conformation. hsp regulates many client proteins involved in signal transduction. other important functions include stimulation of chemotaxis, migration, proliferation and cytokine secretion in malignant and endothelial cells. we show for the first time that hsp is a functionally critical component of the mactinin-binding heterocomplex on monocytic cells. two other proteins of molecular weight and kd found in this complex may possibly represent other hsps, since previous studies have shown that hsp90/ forms a receptor complex in monocytes and macrophages. many hsps interact with components of the actin cytoskeleton, perhaps modulating the cytoskeletal response to stress. it has been reported that hsp and hsp bind actin, while the smaller hsp has been reported to associate with both actin and α-actinin. however, it is likely that the direct binding of extracellular mactinin is to other protein on the surface of hl- cells, which then form a hetero-complex with hsp in monocytes/macrophages and related cells, hsp potently induces tnf-α and il-1β expression and activation and cell cycling and differentiation. the hsp-activated macrophage axis may also play an important role in atherosclerosis. inhibitors of hsp such as geldanamycin and its analogues, have been used to identify hsp substrates. we found that geldanamycin and two of its analogues markedly inhibited mactinin-induced chemotaxis and cytokine production. their ability to inhibit chemotaxis was specific for mactinin since they had no significant effect on fmlp-induced chemotaxis. we also found that hsp inhibitors blocked mactinin-induced cytokine production from monocytes, confirming that hsp mediates the stimulatory activity of mactinin on monocytes. our results suggest that a cytoskeletal protein derivative may propagate inflammation, in part, by stimulating production of the pro-inflammatory cytokines il-1α, il-1β and tnf-α. these two cytokines are elevated in the serum and synovial fluid of patients with rheumatoid arthritis, and both are capable of inducing joint damage in experimental arthritis models. the clinical efficacy of agents that block these cytokines in ameliorating bone and joint destruction in arthritis is now widely accepted. hsp induces urokinase-type plasminogen activator secretion and urokinase secreted by activated monocytes degrades α-actinin to generate mactinin. it is therefore possible that the binding and stimulation of hsp activity on monocytes by mactinin itself may represent a positive feed-forward loop that further amplifies the immune/inflammatory response. we therefore speculate that agents that block mactinin may provide a novel strategy for inhibiting hsp activity in diverse diseases.  we have identified that hsp mediates the stimulation of monocytes by mactinin, a fragment of the cytoskeletal protein α-actinin. mactinin represents a novel inducer of hsp activity on monocytes. our findings suggest that inhibition of mactinin may be of benefit in diseases propagated by monocyte/macrophage activation or hsp activity. 
mactinin is a novel inducer of hsp activity on monocytes and may serve to perpetuate and augment monocytic activation, thereby functioning as a "matrikine." we previously showed that mactinin, a degradative fragment of the cytoskeletal protein α-actinin, is present at sites of monocytic activation in vivo, has chemotactic activity for monocytes and promotes monocyte/macrophage maturation. monocytes, their progeny such as dendritic cells and osteoclasts and products including tumor necrosis factor -α, interleukin -1α and il-1β play important roles in cancer, inflammation, immune response and atherosclerosis. affinity purification, mass spectroscopy and western immunoblotting identified heat shock protein - as the kd component of this complex. hsp was responsible for mediating the functional effects of mactinin on monocytes, since hsp inhibitors -17-demethoxygeldanamycin ) almost completely abrogated the stimulatory activity of mactinin on monocytes.
 monocytes, their progeny such as dendritic cells and osteoclasts and products including tumor necrosis factor -α, interleukin -1α and il-1β play important roles in cancer, inflammation, immune response and atherosclerosis. we previously showed that mactinin, a degradative fragment of the cytoskeletal protein α-actinin, is present at sites of monocytic activation in vivo, has chemotactic activity for monocytes and promotes monocyte/macrophage maturation. we therefore sought to determine the mechanism by which mactinin stimulates monocytes.  radiolabeled mactinin bound to a heterocomplex on monocytes comprised of at least proteins of molecular weight kd, kd and kd. affinity purification, mass spectroscopy and western immunoblotting identified heat shock protein - as the kd component of this complex. hsp was responsible for mediating the functional effects of mactinin on monocytes, since hsp inhibitors -17-demethoxygeldanamycin ) almost completely abrogated the stimulatory activity of mactinin on monocytes.  mactinin is a novel inducer of hsp activity on monocytes and may serve to perpetuate and augment monocytic activation, thereby functioning as a "matrikine." blockage of this function of mactinin may be useful in diseases where monocyte/macrophage activation and/or hsp activity are detrimental. 