BACKGROUND
the mammary gland  secretion is essential for the survival of neonatal mammals  <cit> .
this organ is a tertiary extralymphoid tissue with non-organised lymphoid components  <cit> .
its vasculature and epithelium expand in size during gestation and lactation.
in all mammalian species, this gland is colonised by t and b lymphocytes  <cit> .
t lymphocytes accumulate in the mg during gestation whereas b cells and their plasma cell derivatives progressively accumulate during lactation  <cit> .
colostrum and milk immunoglobulins  are the net result of ig extravasation from blood and local ig production by plasma cells  <cit> .
these plasma cells are derived from remote tissue precursors which are induced either in systemic and in mucosal sites, depending on ig isotype.
in single-stomached animals, iga is secreted by iga plasmablasts, which originate in the gut  <cit>  whereas igg is derived from systemic sites  <cit> .
it has been shown that the cc chemokine ccl <dig>  <cit>  plays a major role in iga plasma cell recruitment.
other factors may also be involved, particularly for other types of plasma cell.
indeed, unidentified factors in milk increase the migration of mouse igg- and iga-bearing mucosal lymphocytes  <cit> .
for example, tumour necrosis factor isolated from milk has been shown to increase the migration of lymphocytes  <cit> .
moreover, other studies have reported that milk contains factors with cell regulatory and stimulatory properties.
these factors include hormones and cytokines, which do not have specific effects on any given lymphocyte subset  <cit> .
we have shown that swine whey has chemoattractant activity  for swine lymphocytes  <cit> .
we therefore hypothesized that whey was involved in the local recruitment of plasma cells  <cit> .
we tested this hypothesis by fractionating sow milk and assessing the ability of purified fractions to increase lymphocyte or b lymphoblast migration in relation to the stage of development of the healthy mammary gland.
RESULTS
chemoattractant activity of whey ultrafiltrate
previous investigations have shown that the removal of fat and casein did not result in the loss of the original ca of skimmed milk .
we show here that the <  <dig> kda ultrafiltrate fraction  gave a bell-shaped dose-response curve typical of chemoattractants at the same optimal dilution , and with a similar number of migrating cells  to unfiltered whey.
on the contrary, the >  <dig> kda fraction was inactive .
lymph node  lymphocytes  and b lymphoblasts  migrated to similar extents.
neutrophils  <cit>  and monocytes  purified from the swine blood do not exhibit any ca with fmlp and the <  <dig> kda ultrafiltrate fraction.
these results combined with morphological data demonstrate that ca activity is restricted to lymphoid cells.
as ca was similar for lymphocytes from the mesenteric lymp node  and the ileal lymph node , we subsequently evaluated ca principally on b-cell lymphoblasts and mln lymphocytes.
rp-hplc fractionation and purification of the milk chemoattractant
in two fractions  of the first rp-hplc fractionation of uf <dig> , ca was recovered at the original dilution , with b lymphoblasts and mln lymphocytes, whereas f <dig> was inactive.
after further fractionation, only the f2a and f2b fractions were active  with both b-cell lymphoblasts and lymphocytes at optimal and suboptimal dilutions, whereas the f1a fraction was only active at optimal dilution.
we fractionated f2a and f2b further and found that only the f2b <dig> subfraction was active with both b-cell lymphoblasts and mln lymphocytes at optimal and suboptimal concentrations, whereas the f2a <dig> and f2a <dig> subfractions were not active at suboptimal concentrations.
the initial ca of uf <dig> was recovered mostly in the f2b <dig> subfraction, which was subjected to a fourth run of rp-hplc, .
ca with both lymphoblasts  and lymphocytes was recovered in the f2b1-a and f2b1-b peaks.
identification of the chemotactic peptide
the two most chemoattractant peaks, f2b1-a and f2b1-b, were characterised by ms .
these peaks were found to correspond to five peptides with overlapping sequences.
a  <dig> da peptide was found in both the a and b peaks, whereas the  <dig> da peptide was present in peak a but not in peak b. the peptides were sequenced by ms-ms and homology searches of databases  indicated that all sequenced peptides in peaks a and b were derived from the same serum amyloid a protein , with the exception of an unidentified  <dig> da peptide.
we assessed if these peptides derived from saa were responsible for the ca of the f2b <dig> fraction, by testing the ca of the synthetic peptides dmreanyknsdkyfhargnydaa  and rppglpdky  separately.
these two synthetic peptides eluted with the same retention times as peaks a and b from f2b <dig> in rp-hplc  under the same conditions .
we also showed that f2b1-a induced potent chemotaxis .
the dilution-effect curve  showed that ca was recovered with the  <dig>  kda peptide, albeit at  <dig> times lower concentrations for lymphoblasts  than for mln lymphocytes , whereas the  <dig>  kda peptide had no activity.
furthermore, there were two peaks of ca with iln lymphocytes  at  <dig>  μm and  <dig>  nm.
at the lower concentration, most of the migrating cells were lymphoblasts, in clusters of  <dig> to  <dig> cells.
this result is consistent with the effects on l <dig> b lymphoblasts, which was observed at the same concentration .
at the higher concentration, the  <dig>  kda peptide induced the migration of small iln lymphocytes, consistent with the observed effects on small mln lymphocytes .
we investigated whether peptides were chemotactic or chemokinetic, using the checkerboard assay  with b lymphoblasts.
maximal migration occurred when higher molar concentrations of the  <dig>  kda peptides were placed in the lower compartment and no significant increase in migration was observed when equal molar concentrations of  <dig>  kda peptides were placed on both sides of the filter, thereby demonstrating that the  <dig>  kda residue peptide was not chemokinetic.
the  <dig> kda peptide did not display any activity potentially attributable to a chemokinetic or chemotactic response.
amigration was assessed with b lymphoblasts after  <dig> h of incubation in the presence of peptides in the upper, lower or both compartments.
for underlined values, there was no gradient of peptide concentration between the upper and the lower compartments.
in each column, above the underlined values, the peptide concentrations were lower than in the upper compartment and below the underlined values, the peptide concentrations were higher than in the upper compartment.
results are means ± sem of migrating cells, for experiments carried out in triplicate.
bspontaneous migration in the absence of peptide.
csignificant difference between migration in the absence of a gradient  and peptide-induced migration .
a polycarbonate filter with  <dig> μm pores separated the two compartments of the chamber.
chemoattractant activity with separated cd3+ and sig+ cells from mesenteric lymph node
cd3+ t lymphocytes and sig+ b cells were purified by immunomagnetic positive sorting.
the enrichment for cd <dig> was > 95% and the depletion for sig > 95% .
the migration of cd3+ t cells in response to uf <dig> did not differ from spontaneous migration , whereas the cd3- fraction induced a dose-dependent increase in migration.
conversely, the extent of migration of the purified sig+ b cells  towards the  <dig> da peptide was higher than that of spontaneous migration, at a concentration of  <dig>  μm , whereas the extent of migration of the sig- fraction did not differ  from that of spontaneous migration.
no increase in migration towards the  <dig> da peptide was  observed in either sig+ b cells or sig-depleted cells.
consequently, the  <dig> da peptide is chemoattractant for swine sig+ b cells, but not for cd3+ t cells.
furthermore, the peptide was not chemoattractant for purified porcine neutrophils and monocytes .
saa mrna production in the porcine mammary gland
we then assessed porcine saa mrna expression in the mg as a function of developmental stage.
mg cdna was amplified by pcr using primers  designed into two relatively well conserved saa regions.
a single pcr product, corresponding to an internal fragment of the porcine saa cdna, was obtained .
its deduced amino-acid sequence carried the signature of all serum amyloid a proteins: argnyda-krg-gg-wa.
moreover, this sequence showed a very high percentage of amino acids identical to those of the saa <dig> sequences of sus scrofa  now available .
as a single sequence was amplified by pcr, this "saa2" is probably the most predominant member of the saa family expressed in the mg. production of the porcine saa mrna in mg was then assessed, using a second primer pair  designed on the basis of our porcine saa cdna sequence.
in porcine mg, saa mrna was detectable during gestation, and its level began to increase significantly at day  <dig> of gestation, reaching a peak at day  <dig> of lactation .
the saa mrna expression was higher in the second half of lactation than at any stage of gestation .
peptide-induced mobilization of intracellular ca++ in differentiated hl- <dig> cells and fprl2-expressing hl- <dig> cells
because the  <dig>  kda peptide  exhibits a high degree of amino acid identity with human saa, which previously has been shown to bind to and to activate the human n-formyl peptide receptor-like  <dig> , we tested whether it could activate a fprl1- or fprl2-dependent calcium response.
fprl <dig> is a member of the fpr family that is expressed in human neutrophils and differentiated hl- <dig> cells as well as monocytes, whereas the n-formyl peptide receptor-like  <dig>  is expressed in human monocytes but not in neutrophils.
when added to differentiated hl- <dig> cells or undifferentiated fprl2-expressing hl- <dig> cells, the  <dig>  kda synthetic peptide  was unable to induce intracellular calcium mobilization.
in contrast, recombinant human aposaa, an agonist specific of fprl <dig>  or wkymvm, an agonist of fprl <dig>  triggered a robust calcium response in differentiated hl- <dig> cells or undifferentiated fprl2-expressing cells, respectively.
this observation indicates that the absence of response with b cells could not be due to a lack of expression of fprl <dig>  or fprl <dig> .
discussion
in this study, most of the ca of milk for b lymphocytes and b lymphoblasts was detected with the  <dig> da peptide isolated from the whey while no ca activity was detected in the  <dig> da peptide from the same fraction.
ca activity was not detected for neutrophils and monocytes, either onto filter or with purified blood cells.
amino-acid sequencing of the two peptides showed that they belonged to the saa protein family  <cit> .
the partial sequence of the saa cdna amplified from the mg suggested that these peptides might be derived from saa <dig>  as the dna sequence obtained was 98% identical to that of porcine saa <dig>  <cit> .
however, we cannot draw definitive conclusions concerning the saa subtype corresponding to the  <dig>  kda chemoattractant peptide, as this peptide displayed a high degree of identity  with saa subtypes from different origins.
our data suggesting that saa <dig> predominates in the mg, are reminiscent of the finding that bovine mammary-saa or saa <dig> differs from serum-aa in being present at extrahepatic sites, such as the gut epithelium, mg epithelium  <cit>  and in colostrum and milk in healthy animals  <cit> .
however, human breast was found to contain saa <dig>  saa <dig> and saa <dig>  indicating that there is considerable variation amongst species, despite the sequence similarities.
in swine saa <dig>  there is no deletion of the  <dig> aa  of the human sequence required for acute inflammation reaction  <cit> .
this observation provides further evidence that this peptide could play a physiological role in normal mg. the catabolic pathway of saa has yet to be completely defined, but various studies have suggested that saa is degraded by cell-associated elastase-like proteases  <cit>  and several other serine proteases found in serum and milk, including kallikrein and plasmin  <cit> .
the proteolysis of saa by these enzymes leads to the generation of specific peptide fragments, some corresponding to amino acids  <dig> to  <dig> of saa sequence, whereas the chemoattractant peptide is longer  and has different cleavage sites.
the specific action of the mammary saa peptide on b lymphoid cells contrasts with the previously described activity of saa.
indeed human saa is described as involved in adhesion induction, migration, and tissue infiltration of t lymphocytes  <cit> .
the  <dig>  kda peptide  is  <dig> % identical to the 29– <dig> peptide of hsaa-a   <cit> , suggesting a similar function.
however, only a few studies have used synthetic peptides: one study reported that hsaa29– <dig> inhibited adhesion  <cit> , whereas another found no inhibitory effect on neutrophil migration  <cit> .
it has been shown that hsaa and the 29– <dig> fragment display agonist activity involving fprl <dig> receptor  <cit> .
moreover, fmlp receptor has not only been described on human neutrophils and nk cells but also on b cells  <cit> .
as in our study, the fact that b cells respond to μmolar concentrations suggest that they exhibit fprl <dig> or fprl <dig> receptors.
however, we may exclude fprl <dig> as this receptor does not bind or respond to n-formyl peptides  <cit> .
on the other hand, swine l <dig> b cells incubated with human aposaa or swine  <dig>  kda peptide failed to exhibit any calcium flux.
these results indicate that the peptide probably uses a receptor other than fprl <dig> and fprl <dig>  this receptor could be the recently described "receptor independent of fprl <dig>  <cit> , which did not lead to a calcium influx.
furthermore, this receptor could be present in two similar forms: one of lower avidity on b cells and the other of higher avidity or subject to up-regulation after the activation of b cells or b lymphoblasts.
alternatively, these results could fit with the observation that b lymphocytes can respond without mobilization of intracellular calcium as observed, for example, with chemokines ccl <dig>  ccl <dig>  cxcl <dig> and cxcl <dig>  <cit> .
the lack of ca of the terminal saa peptide  with b cells does not entirely rule out activity with these cells.
indeed, the truncated form of hsaa, aglpdky , and analogues are known to bind human cd4+ t lymphocytes and to stimulate them to produce interferon-γ.
it is also possible that the 98– <dig> peptide makes the cells susceptible to the 29– <dig> fragment chemoattraction after activation.
binding may also lead to the induction of cellular responses, such as cytokine secretion  <cit>  and the up-regulation of integrin α4β <dig>  in particular.
α4β <dig> expression increases the probability of in vivo binding to vcam- <dig>  <cit> , a vascular addressin present on the mg blood vessels  <cit> .
the lymphocyte aggregation observed in the present study  would be consistent with the expected effect of such binding.
the yigsd laminin-like element of hsaa is replaced by the yknsd element in psaa, and both hsaa and psaa express rgn motif, a "rgd like" motif.
this rgn motif  <cit>  represents integrin-binding and cell attachment motifs derived from cellular adhesion protein molecules, such as fibronectin, collagen, vitronectin, fibrinogen, laminin and many microbial proteins, including the tat protein of the human immunodeficiency virus  <cit> .
binding of the rgn motif to the cell membrane is known to stimulate membrane integrins, leading to a local increase in the concentrations of f-actin and α-actinin, resulting in cell locomotion  <cit> .
it is possible that the peptide adheres to the filter via this rgn motif, inducing lymphocyte migration.
transposed in vivo, these data suggest a similar phenomenon, in which the binding of saa to the surface of the mammary endothelial cells may induce b-cell adhesion followed by the transendothelial migration of these cells in an saa-peptide gradient.
finally, in addition to binding to endothelial cells, saa also binds to the basal membrane and extracellular matrix, probably due to its heparin-binding properties  <cit> .
thus, gradients of extracellular tissue-bound saa or saa peptides, including the  <dig>  kda peptide, may induce haptotactic b-cell migration in the mammary tissue during lactation.
saa mrna and protein productions have been recently demonstrated in extrahepatic tissues, and specifically in mg, mostly in epithelial and endothelial cells  <cit> , supporting the hypothesis of a role in lymphocyte recruitment.
high extrahepatic expressions and the secretion of the mammary-associated serum amyloid a  <dig> isoform  in the colostrum and milk of healthy animals has been reported  <cit> .
although the functional significance of extrahepatic saa production remains unclear, it has been suggested that the apparent constitutive production of the saa transcript in non-hepatic cell lines is consistent with a housekeeping role for the protein  <cit> , either as a first line of defence or in the maintenance of tissue function.
previous findings of the upregulation of saa gene expression in response to prolactin  <cit>  and the increase in b lymphoblast numbers in the mg during lactation  <cit>  would together be consistent with the results of the present study.
the human mg produces and releases bioactive prolactin in situ  <cit> .
in this study, we showed that porcine saa mrna production increased during lactation, consistent with the hormonal regulation of saa production mediated by an increase in prolactin concentration at this stage of mg development.
this milk peptide, due to its small size, could cross the neonatal gut epithelium and stimulate the neonatal gut immune response by enhancing the recruitment of mucosal gut b lymphoblasts.
CONCLUSIONS
in the current paper, we have investigated whether physiological chemoattractants induced the recruitment of immune cells from the maternal bloodstream in lactating mammary glands, by analysing the chemoattractant activity of various milk fractions.
the b cell chemoattractant activity of a saa  <dig>  kda peptide and the concomitant up-regulation of saa mrna during lactation are consistent with the saa23– <dig> fragment being involved in preplasma b-cell recruitment to the mammary gland, potentially influencing ig concentrations, conferring active and passive protection on neonates and providing local protection for the mammary gland.

 the mammary gland secretion is essential for the survival of neonatal mammals. this organ is a tertiary extralymphoid tissue with non-organised lymphoid components. its vasculature and epithelium expand in size during gestation and lactation. in all mammalian species, this gland is colonised by t and b lymphocytes. t lymphocytes accumulate in the mg during gestation whereas b cells and their plasma cell derivatives progressively accumulate during lactation. colostrum and milk immunoglobulins are the net result of ig extravasation from blood and local ig production by plasma cells. these plasma cells are derived from remote tissue precursors which are induced either in systemic and in mucosal sites, depending on ig isotype. in single-stomached animals, iga is secreted by iga plasmablasts, which originate in the gut whereas igg is derived from systemic sites. it has been shown that the cc chemokine ccl plays a major role in iga plasma cell recruitment. other factors may also be involved, particularly for other types of plasma cell. indeed, unidentified factors in milk increase the migration of mouse igg- and iga-bearing mucosal lymphocytes. for example, tumour necrosis factor isolated from milk has been shown to increase the migration of lymphocytes. moreover, other studies have reported that milk contains factors with cell regulatory and stimulatory properties. these factors include hormones and cytokines, which do not have specific effects on any given lymphocyte subset. we have shown that swine whey has chemoattractant activity for swine lymphocytes. we therefore hypothesized that whey was involved in the local recruitment of plasma cells. we tested this hypothesis by fractionating sow milk and assessing the ability of purified fractions to increase lymphocyte or b lymphoblast migration in relation to the stage of development of the healthy mammary gland.  chemoattractant activity of whey ultrafiltrate previous investigations have shown that the removal of fat and casein did not result in the loss of the original ca of skimmed milk. we show here that the < kda ultrafiltrate fraction gave a bell-shaped dose-response curve typical of chemoattractants at the same optimal dilution, and with a similar number of migrating cells to unfiltered whey. on the contrary, the > kda fraction was inactive. lymph node lymphocytes and b lymphoblasts migrated to similar extents. neutrophils and monocytes purified from the swine blood do not exhibit any ca with fmlp and the < kda ultrafiltrate fraction. these results combined with morphological data demonstrate that ca activity is restricted to lymphoid cells. as ca was similar for lymphocytes from the mesenteric lymp node and the ileal lymph node, we subsequently evaluated ca principally on b-cell lymphoblasts and mln lymphocytes. rp-hplc fractionation and purification of the milk chemoattractant in two fractions of the first rp-hplc fractionation of uf, ca was recovered at the original dilution, with b lymphoblasts and mln lymphocytes, whereas f was inactive. after further fractionation, only the f2a and f2b fractions were active with both b-cell lymphoblasts and lymphocytes at optimal and suboptimal dilutions, whereas the f1a fraction was only active at optimal dilution. we fractionated f2a and f2b further and found that only the f2b subfraction was active with both b-cell lymphoblasts and mln lymphocytes at optimal and suboptimal concentrations, whereas the f2a and f2a subfractions were not active at suboptimal concentrations. the initial ca of uf was recovered mostly in the f2b subfraction, which was subjected to a fourth run of rp-hplc,. ca with both lymphoblasts and lymphocytes was recovered in the f2b1-a and f2b1-b peaks. identification of the chemotactic peptide the two most chemoattractant peaks, f2b1-a and f2b1-b, were characterised by ms. these peaks were found to correspond to five peptides with overlapping sequences. a da peptide was found in both the a and b peaks, whereas the da peptide was present in peak a but not in peak b. the peptides were sequenced by ms-ms and homology searches of databases indicated that all sequenced peptides in peaks a and b were derived from the same serum amyloid a protein, with the exception of an unidentified da peptide. we assessed if these peptides derived from saa were responsible for the ca of the f2b fraction, by testing the ca of the synthetic peptides dmreanyknsdkyfhargnydaa and rppglpdky separately. these two synthetic peptides eluted with the same retention times as peaks a and b from f2b in rp-hplc under the same conditions. we also showed that f2b1-a induced potent chemotaxis. the dilution-effect curve showed that ca was recovered with the kda peptide, albeit at times lower concentrations for lymphoblasts than for mln lymphocytes, whereas the kda peptide had no activity. furthermore, there were two peaks of ca with iln lymphocytes at μm and nm. at the lower concentration, most of the migrating cells were lymphoblasts, in clusters of to cells. this result is consistent with the effects on l b lymphoblasts, which was observed at the same concentration. at the higher concentration, the kda peptide induced the migration of small iln lymphocytes, consistent with the observed effects on small mln lymphocytes. we investigated whether peptides were chemotactic or chemokinetic, using the checkerboard assay with b lymphoblasts. maximal migration occurred when higher molar concentrations of the kda peptides were placed in the lower compartment and no significant increase in migration was observed when equal molar concentrations of kda peptides were placed on both sides of the filter, thereby demonstrating that the kda residue peptide was not chemokinetic. the kda peptide did not display any activity potentially attributable to a chemokinetic or chemotactic response. amigration was assessed with b lymphoblasts after h of incubation in the presence of peptides in the upper, lower or both compartments. for underlined values, there was no gradient of peptide concentration between the upper and the lower compartments. in each column, above the underlined values, the peptide concentrations were lower than in the upper compartment and below the underlined values, the peptide concentrations were higher than in the upper compartment. results are means ± sem of migrating cells, for experiments carried out in triplicate. bspontaneous migration in the absence of peptide. csignificant difference between migration in the absence of a gradient and peptide-induced migration. a polycarbonate filter with μm pores separated the two compartments of the chamber. chemoattractant activity with separated cd3+ and sig+ cells from mesenteric lymph node cd3+ t lymphocytes and sig+ b cells were purified by immunomagnetic positive sorting. the enrichment for cd was > 95% and the depletion for sig > 95%. the migration of cd3+ t cells in response to uf did not differ from spontaneous migration, whereas the cd3- fraction induced a dose-dependent increase in migration. conversely, the extent of migration of the purified sig+ b cells towards the da peptide was higher than that of spontaneous migration, at a concentration of μm, whereas the extent of migration of the sig- fraction did not differ from that of spontaneous migration. no increase in migration towards the da peptide was observed in either sig+ b cells or sig-depleted cells. consequently, the da peptide is chemoattractant for swine sig+ b cells, but not for cd3+ t cells. furthermore, the peptide was not chemoattractant for purified porcine neutrophils and monocytes. saa mrna production in the porcine mammary gland we then assessed porcine saa mrna expression in the mg as a function of developmental stage. mg cdna was amplified by pcr using primers designed into two relatively well conserved saa regions. a single pcr product, corresponding to an internal fragment of the porcine saa cdna, was obtained. its deduced amino-acid sequence carried the signature of all serum amyloid a proteins: argnyda-krg-gg-wa. moreover, this sequence showed a very high percentage of amino acids identical to those of the saa sequences of sus scrofa now available. as a single sequence was amplified by pcr, this "saa2" is probably the most predominant member of the saa family expressed in the mg. production of the porcine saa mrna in mg was then assessed, using a second primer pair designed on the basis of our porcine saa cdna sequence. in porcine mg, saa mrna was detectable during gestation, and its level began to increase significantly at day of gestation, reaching a peak at day of lactation. the saa mrna expression was higher in the second half of lactation than at any stage of gestation. peptide-induced mobilization of intracellular ca++ in differentiated hl- cells and fprl2-expressing hl- cells because the kda peptide exhibits a high degree of amino acid identity with human saa, which previously has been shown to bind to and to activate the human n-formyl peptide receptor-like, we tested whether it could activate a fprl1- or fprl2-dependent calcium response. fprl is a member of the fpr family that is expressed in human neutrophils and differentiated hl- cells as well as monocytes, whereas the n-formyl peptide receptor-like is expressed in human monocytes but not in neutrophils. when added to differentiated hl- cells or undifferentiated fprl2-expressing hl- cells, the kda synthetic peptide was unable to induce intracellular calcium mobilization. in contrast, recombinant human aposaa, an agonist specific of fprl or wkymvm, an agonist of fprl triggered a robust calcium response in differentiated hl- cells or undifferentiated fprl2-expressing cells, respectively. this observation indicates that the absence of response with b cells could not be due to a lack of expression of fprl or fprl. discussion in this study, most of the ca of milk for b lymphocytes and b lymphoblasts was detected with the da peptide isolated from the whey while no ca activity was detected in the da peptide from the same fraction. ca activity was not detected for neutrophils and monocytes, either onto filter or with purified blood cells. amino-acid sequencing of the two peptides showed that they belonged to the saa protein family. the partial sequence of the saa cdna amplified from the mg suggested that these peptides might be derived from saa as the dna sequence obtained was 98% identical to that of porcine saa. however, we cannot draw definitive conclusions concerning the saa subtype corresponding to the kda chemoattractant peptide, as this peptide displayed a high degree of identity with saa subtypes from different origins. our data suggesting that saa predominates in the mg, are reminiscent of the finding that bovine mammary-saa or saa differs from serum-aa in being present at extrahepatic sites, such as the gut epithelium, mg epithelium and in colostrum and milk in healthy animals. however, human breast was found to contain saa saa and saa indicating that there is considerable variation amongst species, despite the sequence similarities. in swine saa there is no deletion of the aa of the human sequence required for acute inflammation reaction. this observation provides further evidence that this peptide could play a physiological role in normal mg. the catabolic pathway of saa has yet to be completely defined, but various studies have suggested that saa is degraded by cell-associated elastase-like proteases and several other serine proteases found in serum and milk, including kallikrein and plasmin. the proteolysis of saa by these enzymes leads to the generation of specific peptide fragments, some corresponding to amino acids to of saa sequence, whereas the chemoattractant peptide is longer and has different cleavage sites. the specific action of the mammary saa peptide on b lymphoid cells contrasts with the previously described activity of saa. indeed human saa is described as involved in adhesion induction, migration, and tissue infiltration of t lymphocytes. the kda peptide is % identical to the 29– peptide of hsaa-a, suggesting a similar function. however, only a few studies have used synthetic peptides: one study reported that hsaa29– inhibited adhesion, whereas another found no inhibitory effect on neutrophil migration. it has been shown that hsaa and the 29– fragment display agonist activity involving fprl receptor. moreover, fmlp receptor has not only been described on human neutrophils and nk cells but also on b cells. as in our study, the fact that b cells respond to μmolar concentrations suggest that they exhibit fprl or fprl receptors. however, we may exclude fprl as this receptor does not bind or respond to n-formyl peptides. on the other hand, swine l b cells incubated with human aposaa or swine kda peptide failed to exhibit any calcium flux. these results indicate that the peptide probably uses a receptor other than fprl and fprl this receptor could be the recently described "receptor independent of fprl, which did not lead to a calcium influx. furthermore, this receptor could be present in two similar forms: one of lower avidity on b cells and the other of higher avidity or subject to up-regulation after the activation of b cells or b lymphoblasts. alternatively, these results could fit with the observation that b lymphocytes can respond without mobilization of intracellular calcium as observed, for example, with chemokines ccl ccl cxcl and cxcl. the lack of ca of the terminal saa peptide with b cells does not entirely rule out activity with these cells. indeed, the truncated form of hsaa, aglpdky, and analogues are known to bind human cd4+ t lymphocytes and to stimulate them to produce interferon-γ. it is also possible that the 98– peptide makes the cells susceptible to the 29– fragment chemoattraction after activation. binding may also lead to the induction of cellular responses, such as cytokine secretion and the up-regulation of integrin α4β in particular. α4β expression increases the probability of in vivo binding to vcam-, a vascular addressin present on the mg blood vessels. the lymphocyte aggregation observed in the present study would be consistent with the expected effect of such binding. the yigsd laminin-like element of hsaa is replaced by the yknsd element in psaa, and both hsaa and psaa express rgn motif, a "rgd like" motif. this rgn motif represents integrin-binding and cell attachment motifs derived from cellular adhesion protein molecules, such as fibronectin, collagen, vitronectin, fibrinogen, laminin and many microbial proteins, including the tat protein of the human immunodeficiency virus. binding of the rgn motif to the cell membrane is known to stimulate membrane integrins, leading to a local increase in the concentrations of f-actin and α-actinin, resulting in cell locomotion. it is possible that the peptide adheres to the filter via this rgn motif, inducing lymphocyte migration. transposed in vivo, these data suggest a similar phenomenon, in which the binding of saa to the surface of the mammary endothelial cells may induce b-cell adhesion followed by the transendothelial migration of these cells in an saa-peptide gradient. finally, in addition to binding to endothelial cells, saa also binds to the basal membrane and extracellular matrix, probably due to its heparin-binding properties. thus, gradients of extracellular tissue-bound saa or saa peptides, including the kda peptide, may induce haptotactic b-cell migration in the mammary tissue during lactation. saa mrna and protein productions have been recently demonstrated in extrahepatic tissues, and specifically in mg, mostly in epithelial and endothelial cells, supporting the hypothesis of a role in lymphocyte recruitment. high extrahepatic expressions and the secretion of the mammary-associated serum amyloid a isoform in the colostrum and milk of healthy animals has been reported. although the functional significance of extrahepatic saa production remains unclear, it has been suggested that the apparent constitutive production of the saa transcript in non-hepatic cell lines is consistent with a housekeeping role for the protein, either as a first line of defence or in the maintenance of tissue function. previous findings of the upregulation of saa gene expression in response to prolactin and the increase in b lymphoblast numbers in the mg during lactation would together be consistent with the results of the present study. the human mg produces and releases bioactive prolactin in situ. in this study, we showed that porcine saa mrna production increased during lactation, consistent with the hormonal regulation of saa production mediated by an increase in prolactin concentration at this stage of mg development. this milk peptide, due to its small size, could cross the neonatal gut epithelium and stimulate the neonatal gut immune response by enhancing the recruitment of mucosal gut b lymphoblasts.  in the current paper, we have investigated whether physiological chemoattractants induced the recruitment of immune cells from the maternal bloodstream in lactating mammary glands, by analysing the chemoattractant activity of various milk fractions. the b cell chemoattractant activity of a saa kda peptide and the concomitant up-regulation of saa mrna during lactation are consistent with the saa23– fragment being involved in preplasma b-cell recruitment to the mammary gland, potentially influencing ig concentrations, conferring active and passive protection on neonates and providing local protection for the mammary gland. 
milk chemoattractants are potentially involved in the recruitment of lymphocytes from the maternal bloodstream in lactating mammary glands. normal mammary gland contains an extravascular population of b lymphoblasts, precursors of the immunoglobulin plasma cells that play a key role in the passive protection of neonates by secreting immunoglobulins to colostrum and milk. these data are consistent with the saa23– fragment being involved in preplasma b-cell recruitment to the mammary gland and resultant benefit to the neonate. then, the saa mrna expression was analysed and we observed more saa transcripts during lactation than gestation. at the lower concentration, it attracted b-cell lymphoblasts, whereas at the higher, it attracted b lymphocytes. we investigated the presence of chemoattractants in the milk by analysing the chemoattractant activity of various fractions of this secretion.
 normal mammary gland contains an extravascular population of b lymphoblasts, precursors of the immunoglobulin plasma cells that play a key role in the passive protection of neonates by secreting immunoglobulins to colostrum and milk. we investigated the presence of chemoattractants in the milk by analysing the chemoattractant activity of various fractions of this secretion. milk chemoattractants are potentially involved in the recruitment of lymphocytes from the maternal bloodstream in lactating mammary glands.  the dilution-related lymphoid cell chemoattraction of whey was associated with a < kda ultrafiltrate. active fractions were purified by reverse-phase high performance liquid chromatography. two peptides of kda and kda were identified as fragments of the saa protein family, tentatively identified as saa only the kda synthetic peptide displayed chemotactic activity, at two different optimal concentrations. at the lower concentration, it attracted b-cell lymphoblasts, whereas at the higher, it attracted b lymphocytes. then, the saa mrna expression was analysed and we observed more saa transcripts during lactation than gestation.  these data are consistent with the saa23– fragment being involved in preplasma b-cell recruitment to the mammary gland and resultant benefit to the neonate. 