BACKGROUND
human mast cells  originate from bone marrow-derived cd34+ pluripotent progenitor cells  <cit>  and migrate as immature cells from the bone marrow to tissue sites including the lung and gastrointestinal tract.
there they mature and participate in both innate and acquired immune responses with production of cytokines and other inflammatory mediators  <cit> .
mast cell growth and development thus may occur in a tissue that interfaces with the external environment, potentially exposing humc during their development to bacterial products which could have an impact on their subsequent behavior.
consistent with this idea is the observation that humc have been shown to express toll-like receptors  1– <dig>  and  <dig> both in vitro and in vivo   <cit> ; and exposure to such bacterial products as endotoxin  or peptidoglycan  leads to expression and release of tnf-α, gm-csf, il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig>  <cit> .
however, and in a related question, it is currently not known whether bacteria-derived products alter the growth and development of humc.
we therefore asked whether long or short-term exposure to lps and pgn could alter specific humc characteristics including growth; surface fcεri and cd <dig> expression; and β-hexosaminidase, ltc <dig> and pgd <dig> release; protease expression and composition, and cytokine release.
as will be shown, lps augmented protease expression and altered protease composition in developing and mature humc, while reducing fcεri expression and ige-mediated degranulation.
this effect in short-term cultures was mediated through humc release of il-1β and il- <dig>  pgn inhibited humc growth at all concentrations studied.
RESULTS
effect of lps and pgn on progenitor humc in  <dig> wk  cultures
to determine the effect of lps or pgn over  <dig> wks on humc growth and development, 10– <dig> ng/ml lps or 10– <dig> μg/ml pgn was added to cd34+ cultures containing  <dig> ng/ml rhscf.
kimura's staining of metachromatic positive humc correlated with tryptase staining of humc granules on cytopreparations, did not differ by greater than 5%, and was used for counting small numbers of humcs available to study, with minimal cell loss.
as shown in figure 1a, incubation of cd34+ cells with scf alone resulted in increased total cell numbers by  <dig> wks which then gradually declined by  <dig> wks, while humc numbers rose steadily from 2– <dig> wks and were sustained up to  <dig> wks, by which time all cells in culture were humc.
the addition of lps to cd34+ cells over  <dig> wks of culture had no effect on total cell or humc numbers .
in contrast, pgn initially increased total cell numbers by 1– <dig> wks, but this increase was short lived, and total numbers rapidly declined after 2– <dig> wks .
humc growth was totally suppressed by pgn at  <dig> μg/ml while pgn at  <dig> μg/ml suppressed humc growth by  <dig> wks.
humc were seen in culture in the presence of  <dig> μg/ml pgn, but numbers were less than half of numbers seen with rhscf alone .
inhibition of humc growth was also seen with another tlr <dig> agonist, lipoteichoic acid .
as seen in figure 1d, lta at  <dig> – <dig> μg/ml decreased total cell numbers starting at 2– <dig> wks.
the addition of lta at  <dig> – <dig> μg/ml suppressed humc growth beginning at 3– <dig> wks and for the duration of the cultures .
thus, the addition of lps over  <dig> wks did not alter humc numbers while pgn and lta inhibited humc outgrowth.
effect of lps and pgn on  <dig> wk old humc cd <dig> and fcεri expression in  <dig> hr  cultures
we next reasoned that exposure to lps or pgn would occur to more mature mast cells in some situations and promote humc differentiation.
we thus cultured mature  <dig> wk old humc in either  <dig> ng/ml or  <dig> ng/ml rhscf in the presence or absence of  <dig> ng/ml lps or  <dig> μg/ml pgn  for  <dig> hrs.
cells were then counted, harvested and examined by flow cytometry for fcεri and cd <dig> expression.
lps  <dig> ng/ml or  <dig> ng/ml pgn had no effect on humc numbers when cultured in combination with rhscf  <dig> ng/ml for  <dig> hr.
when rhscf was reduced to  <dig> ng/ml , humc numbers decreased consistent with less rhscf in the cultures.
viability of the remaining humc after  <dig> hrs ranged from 90–95%.
the added presence of lps increased humc numbers after  <dig> hrs as compared with rhscf alone , while pgn again resulted in fewer humc in culture .
the addition of either  <dig> μg/ml pgn or  <dig> μg/ml lta caused an increase of approximately 27% and 55%, respectively, of apoptotic cells compared to cultures in  <dig> ng/ml rhscf alone, in contrast to  <dig> ng/ml lps which reduced apoptosis by approximately 50%.
to eliminate cell debris or any monocytes from being analyzed, cd117+/fcεri+ humc were gated, followed by examination for other surface markers or intracellular proteases.
as shown in figure 2b, in the presence of  <dig> ng/ml rhscf, a homogeneous population of cd117+/fcεri+ humc was seen .
in the added presence of  <dig> ng/ml lps, cd117+/fcεri+ cells decreased by approximately 7% for humc, with a concurrent decrease in fcεri expression .
reducing the concentration of rhscf to  <dig> ng/ml for  <dig> hrs  gave rise to two distinct humc cd117+ subpopulations: cd117low  and cd117high , with a log-fold increase in mfi for cd117high cells as compared with cultures using  <dig> ng/ml rhscf alone .
the addition of  <dig> ng/ml lps over  <dig> hrs to cd117low and cd117high humc maintained in  <dig> ng/ml rhscf decreased fcεri expression on cd117+/fcεri+ cells  by approximately 9%.
histochemical examination of cd117high and cd117low cells confirmed that both populations shared characteristics, such as numerous surface projections, large and irregular nuclei, and abundant cytoplasmic granules, consistent with cultured humc, as described  <cit> .
thus, fcεri+ expression on humc was downregulated by the presence of lps, while pgn in the presence of  <dig> ng/ml rhscf again inhibited humc numbers.
due to the inhibition of humc growth and development by tlr <dig> agonists pgn and lta, the remaining experiments focused on lps and its humc growth and survival enhancing effects.
effect of lps on  <dig> wk old humc β-hex, pgd <dig> and ltc <dig> release in short-term cultures
we next proceeded to characterize the effect of lps over  <dig> hr on humc, which had been in culture for  <dig> wks, as to histamine content; and β-hex, pgd <dig> and ltc <dig> release.
in the presence of  <dig> ng/ml rhscf, the addition of  <dig> ng/ml lps increased the histamine content of humc over  <dig> hrs from a mean of  <dig>  ±  <dig>  pg/cell to  <dig>  ±  <dig>  pg/cell .
we next assayed release and found that the addition of  <dig> ng/ml lps to  <dig> ng/ml rhscf over  <dig> hrs decreased β-hex release following fcεri crosslinking from  <dig>  ±  <dig> % to  <dig>  ±  <dig> % .
following fcεri crosslinking, lps did not alter pgd <dig> and ltc <dig> release .
thus, the addition of lps to humc cultured over  <dig> hr with  <dig> ng/ml rhscf reduced % β-hex release, but had no effect on pgd <dig> and ltc <dig> release.
effect of lps on  <dig> wk humc protease expression and composition in short-term cultures
we next examined the effect of lps on  <dig> wk old humc protease expression and composition over  <dig> hrs.
in the presence of  <dig> ng/ml rhscf and  <dig> ng/ml lps, facs analysis showed significant increases in tryptase+, chymase+, carboxypeptidase+, tryptase+/chymase+ and tryptase+/carboxypeptidase+ humcs   compared to cultures with  <dig> ng/ml rhscf only.
this effect was not observed when cells were cultured in  <dig> ng/ml of rhscf .
as shown in figure 3b, live gate analysis of the cd117low  and cd117high  humc subpopulations, cultured in the presence of  <dig> ng/ml rhscf with and without  <dig> ng/ml lps, determined that increases in chymase and carboxypeptidase staining were detected predominately for the cd117low subpopulation.
effect of lps and pgn on  <dig> wk old humc cytokine release and the effect of cytokines on protease expression
since mature  <dig> wk humc cultures do not contain other cell types, we next asked whether the effect of lps on protease expression might be induced through endogenous humc cytokine release.
to this end, cell culture supernatants harvested from humc cultures following  <dig> hr incubation with  <dig> or  <dig> ng/ml rhscf alone, or with  <dig> ng/ml lps were assayed for the presence of il-1β, il- <dig>  il- <dig>  il- <dig>  il- <dig>  il- <dig>  il- <dig>  ifn-γ, scf and tnf-α.
as shown in figure 4a,  <dig> ng/ml lps induced humc to produce il-1β, il- <dig>  il- <dig> and il- <dig>  with no differences noted using  <dig> or  <dig> ng/ml rhscf .
no elevations in il- <dig>  il- <dig>  il- <dig>  ifn-γ and tnf-α were noted in the presence of lps.
as expected, scf was detected in cultures containing scf and lps, but levels were not elevated above those measured in cultures with scf alone.
in addition, no elevations in cytokines were noted in cultures to which pgn had been added.
to determine if observed increases in tryptase, chymase and carboxypeptidase expression could be explained on the basis of endogenous il-1β, il- <dig>  il- <dig> and il- <dig> release, humc were cultured with rhscf  <dig> or  <dig> ng/ml alone or with rhil-1β, rhil- <dig>  rhil- <dig> and rhil- <dig> individually or as a four-cytokine mixture, using concentrations of cytokines detected by immunoassay in the culture supernatants .
the combination of  <dig> ng/ml rhscf with either il-1β or il- <dig> gave rise to significant increases in tryptase+, chymase+, tryptase+/chymase+ and tryptase+/carboxypeptidase+ humcs, in contrast to rhil- <dig> or rhil- <dig> which did not increase tryptase, chymase and carboxypeptidase expression .
no increases in protease expression were observed in the presence of  <dig> ng/ml rhscf with other cytokines.
a combination of  <dig> ng/ml rhscf with  <dig> pg/ml rhil-1β,  <dig> pg/ml rhil- <dig>   <dig> pg/ml rhil- <dig> and  <dig> pg/ml rhil- <dig>  cytokine concentrations detected in the cell cultures supernatant following  <dig> hr of incubation with  <dig> ng/ml lps, gave rise to increased tryptase+, chymase+, carboxypeptidase+ , tryptase+/chymase+ and tryptase+/carboxypeptidase+  humc numbers, reproducing the observed increases seen with  <dig> ng/ml lps and  <dig> ng/ml scf .
furthermore, this effect of lps could be significantly reduced when humc were cultured for  <dig> hr in the added presence of  <dig>  μg/ml anti-il-1β and  <dig>  μg/ml anti-il- <dig> antibodies .
qpcr analysis of humc cells showed an overall 8-fold increase in tryptase and 20-fold increase in chymase expression, following stimulation for  <dig> hr with  <dig> ng/ml rhscf,  <dig> pg/ml rhil-1β,  <dig> pg/ml rhil- <dig>   <dig> pg/ml rhil- <dig> and  <dig> pg/ml rhil- <dig> .
similar increases in tryptase and chymase expression were noted following stimulation for  <dig> hr with  <dig> ng/ml rhscf and  <dig> ng/ml lps .
together, this data is consistent with the conclusion that lps stimulates humc in culture to release il-1β, il- <dig>  il- <dig> and il- <dig>  and the overall net effect is to increase cd117low humc production of tryptase, chymase and carboxypeptidase.
effect of lps on 4– <dig> wk old wt and tlr <dig> ko bmmc protease mrna expression in short-term culture
since short-term exposure to lps increased protease expression in humc, we asked whether short-term exposure to lps could also effect the protease mrna expression of bmmc from wt  mice.
thus, bmmc were first allowed to mature over 4– <dig> wks, washed and cultured in either  <dig> or  <dig> ng/ml rmil- <dig>   <dig> or  <dig> ng/ml rmscf alone and with  <dig> ng/ml lps for  <dig> hr.
bmmc were then harvested and examined by qpcr for mmcp mrna expression.
wt bmmc had constitutive mrna expression for the proteases mmcp- <dig>  - <dig>  - <dig> and cpa <dig> when cultured in either rmil- <dig>  or rmscf  for  <dig> hr .
as shown in figure  <dig>  following the addition of lps for  <dig> hr, an upregulation in mmcp- <dig>  mmcp- <dig> and mmcp- <dig> mrna expression was observed in the presence of either rmil <dig>   or in the presence of rmscf  .
a similar pattern of mmcp upregulation was also observed at the higher concentrations of rmil <dig>  or rmscf  in the presence of lps .
since tlr <dig> deficient mice are hyporesponsive to lipopolysaccharide  <cit> , and to determine if upregulated mmcp levels by lps were mediated by tlr <dig>  we obtained bmmc from wt c3h/heouj and tlr <dig> ko c3h/hej mice and exposed them to low concentrations of cytokine in combination with lps.
as can be seen, in the presence of either rmil- <dig>   or rmscf  , wt bmmc from c3h/heouj mice had a similar mmcp mrna profile in response to lps as wt bmmc from c57bl/ <dig> mice.
however, in contrast with c3h/heouj, bmmc cultured from c3h/hej mice and incubated with mil- <dig>  , or rmscf   had reduced mmcp- <dig>  mmcp- <dig> and mmcp- <dig> mrna expression following incubation with lps.
bmmc from both wt and tlr <dig> ko mice were also cultured in the presence of rmil- <dig>  or rmscf  with pgn or lta for  <dig> hr, and examined for any effect on protease mrna expression.
as shown in figures 5e–h, neither tlr <dig> ligand affected mmcp expression in wt or tlr <dig> ko mice.
taken together these data suggest that lps, but not the tlr <dig> ligands pgn or lta, induce the upregulation of mmcp- <dig>  - <dig> and - <dig> mrna expression in bmmc through cytokines which are produced by mast cell activation through tlr <dig>
discussion
in this report, we demonstrate that lps and pgn have very different and specific effects on progenitor and mature humc.
these observations are relevant, since humc maturation may occur in proximity to tissues such as the lung and gastrointestinal mucosa that interface with the external environment, where humc progenitors and their mature counterparts may variably be exposed to bacterial-derived products over time.
it has been shown, for example, that inhalation of aerosolized lps exacerbates allergic airway inflammation in the mouse experimental asthma model through tlr4-mediated mast cell activation and promotion of th <dig> responses  <cit> .
cell transfer experiments using tlr <dig> ko mast cells confirmed that the effect of lps is mediated through tlr <dig> on mast cells, consistent with our data.
we also found that humcs in the presence of lps synthesized de novo il-1β and il- <dig>  in addition to il- <dig> and il- <dig>  which is consistent with the induction of th <dig> effector responses reported for mouse dendritic cell  cultures  <cit> .
if the hypothesis put forward that, under experimental conditions, toll-mediated events facilitate the generation of th <dig> responses  <cit> , then mouse and human in vitro mast cell data are in agreement, and in vivo inhalation of aerosolized lps might induce both humc and dc to produce increased cytokine levels that lead to the promotion of th1-skewed responses.
the net effect of these th <dig> responses in combination with lps-induced th <dig> responses would determine the overall extent of allergic airway inflammation.
we thus examined cd34+-derived humc progenitors over  <dig> wks  and  <dig> wk mature humc over  <dig> hr  for the effect of lps, pgn and lta on cell numbers; and cd <dig>  fcεri, cytokine, tryptase, chymase and carboxypeptidase expression.
in continuous culture over  <dig> wks, lps had little or no effect on humc numbers, while pgn led to a decrease in humc numbers at all concentrations .
lta, another tlr <dig> agonist, also decreased humc numbers at the highest concentrations.
lps, but not pgn, led to small increases in cd <dig>  tryptase and chymase expression compared with rhscf alone, while decreasing fcεri expression .
these differential effects of lps and pgn became more evident when mature  <dig> wk humc were used in short-term cultures.
when  <dig> wk humc, originating from cd34+ cells in rhscf, were cultured with pgn and  <dig> ng/ml rhscf for  <dig> hr, pgn reduced humc numbers below those observed with  <dig> ng/ml rhscf alone.
pgn had no effect on humc numbers when  <dig> ng/ml rhscf was used.
lps somewhat enhanced humc numbers in the presence of  <dig> ng/ml rhscf.
fcεri expression was again reduced in cultures containing either  <dig> or  <dig> ng/ml rhscf and lps , consistent with the observation that the lta, following ligation with tlr <dig>  reduced surface expression of fcεri in isolated human pulmonary mast cells  <cit> .
in the presence of  <dig> ng/ml rhscf, the reduction in fcεri expression could be seen to affect two cd <dig> subpopulations: cd117low and cd117high humcs .
the mfi of the cd117high cells increased a log-fold higher, suggesting that  <dig> ng/ml of rhscf suppressed cd <dig> expression on humc, consistent with other reports  <cit> .
in the presence of lps and  <dig> ng/ml, but not  <dig> ng/ml of rhscf, tryptase positive humc co-expressing chymase and carboxypeptidase increased , and live gate analysis showed that increases in tryptase, chymase and carboxypeptidase occurred predominately in the cd117low subpopulation .
these data therefore demonstrate that short-term exposure of mature humc to varying concentrations of rhscf alters cd <dig> expression  <cit> , and protease expression; and that these effects are modulated by lps.
of note is that  <dig> ng/ml rhscf that we and others use for optimal humc in vitro growth may mask the upregulatory effects of lps, when lps is added to cultures.
it has been reported that activation of murine mast  <cit>  and dendritic  <cit>  cells through tlrs augments production of inflammatory cytokines, and that cytokines can influence human mast cell protease expression  <cit> .
to determine whether lps-driven short-term cultures induced humc-derived cytokine production which in turn could influence cell survival as well as changes in surface receptor and protease expression, we assayed short-term cultures and found, as noted above, significant levels of il-1β, il- <dig>  il- <dig> and il- <dig> in lps-driven, but not pgn-driven, cultures.
furthermore, we were able to reproduce the effects of lps on humc by culturing these cells in  <dig> ng/ml rhscf and immunoassay-detected concentrations of il-1β, il- <dig>  il- <dig> and il- <dig> .
the combination of  <dig> ng/ml rhscf with either il-1β or il- <dig>  but not il- <dig> or il- <dig>  also reproduced the lps observed effects in short-term cultures.
thus, one possible mechanism of action of lps is to stimulate production of mature humc-derived il-1β and il- <dig>  which together promote humc survival, alter humc fcεri and protease expression, and together with il- <dig> direct humc involvement in resolving innate immunity and promoting acquired immune responses.
this transition from innate to acquired immunity involves the resolution of an inflammatory event, and as demonstrated in mouse models, il- <dig> is involved in reducing bacteria-induced inflammation  <cit> .
in addition, although il- <dig> was not shown to affect humc survival, surface receptor and protease expression, il- <dig> production by humc might in part explain how lps can promote neutrophil influx into inflammatory sites and promote host defense mechanisms.
the conclusion that lps upregulates humc protease expression and composition were further supported using bmmc from wt and tlr <dig> ko mice .
it is known that exposure of wt bmmc to rmil <dig> and rmscf alters the protease composition of these cells  <cit> .
following exposure of wt bmmc to reduced concentrations of rmil- <dig> or rmscf in the presence of lps an increase in mrna for the mmcp- <dig>  - <dig> and - <dig> was observed.
bmmc from tlr <dig> ko mice, following stimulation with lps, failed to show any alteration in mmcp- <dig>  mmcp- <dig>  and mmcp- <dig> mrna expression compared with wt controls.
these findings demonstrate that the differential mrna expression of mmcp depends on the presence and concentration of rmil- <dig> or rmscf used to culture bmmc, and that the mmcp mrna expression of bmmc can be influenced by lps ligation with tlr <dig>  an effect which is eliminated in the absence of a functional tlr <dig> receptor.
mmcp- <dig> mrna expression in mucosal mast cells of trichinella spiralis-infected mice can similarly be influenced by il- <dig>  <cit> .
furthermore, mmcp- <dig>  mmcp- <dig> and mmcp- <dig> are chymases, and mmcp- <dig> most probably represents the murine homolog of human chymase  <cit> .
in addition, the observation that tlr <dig> ligands pgn and lta had no effect on mmcp mrna expression from wt and tlr <dig> ko-derived bmmc appears to be consistent with the observation in humc  that lps, but not pgn, increases humc numbers and protease expression, and may be related to the lps-induced increase in il-1β and il- <dig>
the upregulation of human chymase and carboxypeptidase by lps may be another means by which mast cells participate in endotoxin-driven alteration in th <dig> and th <dig> responses.
mast cell-derived chymase and carboxypeptidase are involved in resistance to toxic endogenous or exogenous peptides such as endothelin- <dig>  <cit>  and venoms  <cit>  respectively; thus, their induction may counterbalance the effect of tryptase which is known to increase t cell-derived il- <dig> and airway hyper-responsiveness  <cit> .
CONCLUSIONS
taken together, these data demonstrate that lps can alter humc numbers, influence cytokine and protease expression and composition, and that the effect of lps is best observed when reduced concentrations of rhscf are used, as may occur in tissues.
these data further support the idea that the appearance of lps during a gram negative bacterial infection, in the presence of tissue concentrations of scf, may induce tissue mast cells to express a unique composition of proteases beneficial for controlling and eliminating this particular infection.
in other infections or clinical states having higher concentrations of other cytokines and/or the presence of other mediators, tissue mast cell progenitors may differentially develop and express specific surface receptors and proteases which enable these mast cells to more effectively participate in innate immune responses.

 human mast cells originate from bone marrow-derived cd34+ pluripotent progenitor cells and migrate as immature cells from the bone marrow to tissue sites including the lung and gastrointestinal tract. there they mature and participate in both innate and acquired immune responses with production of cytokines and other inflammatory mediators. mast cell growth and development thus may occur in a tissue that interfaces with the external environment, potentially exposing humc during their development to bacterial products which could have an impact on their subsequent behavior. consistent with this idea is the observation that humc have been shown to express toll-like receptors 1– and both in vitro and in vivo; and exposure to such bacterial products as endotoxin or peptidoglycan leads to expression and release of tnf-α, gm-csf, il- il- il- il- and il-. however, and in a related question, it is currently not known whether bacteria-derived products alter the growth and development of humc. we therefore asked whether long or short-term exposure to lps and pgn could alter specific humc characteristics including growth; surface fcεri and cd expression; and β-hexosaminidase, ltc and pgd release; protease expression and composition, and cytokine release. as will be shown, lps augmented protease expression and altered protease composition in developing and mature humc, while reducing fcεri expression and ige-mediated degranulation. this effect in short-term cultures was mediated through humc release of il-1β and il- pgn inhibited humc growth at all concentrations studied.  effect of lps and pgn on progenitor humc in wk cultures to determine the effect of lps or pgn over wks on humc growth and development, 10– ng/ml lps or 10– μg/ml pgn was added to cd34+ cultures containing ng/ml rhscf. kimura's staining of metachromatic positive humc correlated with tryptase staining of humc granules on cytopreparations, did not differ by greater than 5%, and was used for counting small numbers of humcs available to study, with minimal cell loss. as shown in figure 1a, incubation of cd34+ cells with scf alone resulted in increased total cell numbers by wks which then gradually declined by wks, while humc numbers rose steadily from 2– wks and were sustained up to wks, by which time all cells in culture were humc. the addition of lps to cd34+ cells over wks of culture had no effect on total cell or humc numbers. in contrast, pgn initially increased total cell numbers by 1– wks, but this increase was short lived, and total numbers rapidly declined after 2– wks. humc growth was totally suppressed by pgn at μg/ml while pgn at μg/ml suppressed humc growth by wks. humc were seen in culture in the presence of μg/ml pgn, but numbers were less than half of numbers seen with rhscf alone. inhibition of humc growth was also seen with another tlr agonist, lipoteichoic acid. as seen in figure 1d, lta at – μg/ml decreased total cell numbers starting at 2– wks. the addition of lta at – μg/ml suppressed humc growth beginning at 3– wks and for the duration of the cultures. thus, the addition of lps over wks did not alter humc numbers while pgn and lta inhibited humc outgrowth. effect of lps and pgn on wk old humc cd and fcεri expression in hr cultures we next reasoned that exposure to lps or pgn would occur to more mature mast cells in some situations and promote humc differentiation. we thus cultured mature wk old humc in either ng/ml or ng/ml rhscf in the presence or absence of ng/ml lps or μg/ml pgn for hrs. cells were then counted, harvested and examined by flow cytometry for fcεri and cd expression. lps ng/ml or ng/ml pgn had no effect on humc numbers when cultured in combination with rhscf ng/ml for hr. when rhscf was reduced to ng/ml, humc numbers decreased consistent with less rhscf in the cultures. viability of the remaining humc after hrs ranged from 90–95%. the added presence of lps increased humc numbers after hrs as compared with rhscf alone, while pgn again resulted in fewer humc in culture. the addition of either μg/ml pgn or μg/ml lta caused an increase of approximately 27% and 55%, respectively, of apoptotic cells compared to cultures in ng/ml rhscf alone, in contrast to ng/ml lps which reduced apoptosis by approximately 50%. to eliminate cell debris or any monocytes from being analyzed, cd117+/fcεri+ humc were gated, followed by examination for other surface markers or intracellular proteases. as shown in figure 2b, in the presence of ng/ml rhscf, a homogeneous population of cd117+/fcεri+ humc was seen. in the added presence of ng/ml lps, cd117+/fcεri+ cells decreased by approximately 7% for humc, with a concurrent decrease in fcεri expression. reducing the concentration of rhscf to ng/ml for hrs gave rise to two distinct humc cd117+ subpopulations: cd117low and cd117high, with a log-fold increase in mfi for cd117high cells as compared with cultures using ng/ml rhscf alone. the addition of ng/ml lps over hrs to cd117low and cd117high humc maintained in ng/ml rhscf decreased fcεri expression on cd117+/fcεri+ cells by approximately 9%. histochemical examination of cd117high and cd117low cells confirmed that both populations shared characteristics, such as numerous surface projections, large and irregular nuclei, and abundant cytoplasmic granules, consistent with cultured humc, as described. thus, fcεri+ expression on humc was downregulated by the presence of lps, while pgn in the presence of ng/ml rhscf again inhibited humc numbers. due to the inhibition of humc growth and development by tlr agonists pgn and lta, the remaining experiments focused on lps and its humc growth and survival enhancing effects. effect of lps on wk old humc β-hex, pgd and ltc release in short-term cultures we next proceeded to characterize the effect of lps over hr on humc, which had been in culture for wks, as to histamine content; and β-hex, pgd and ltc release. in the presence of ng/ml rhscf, the addition of ng/ml lps increased the histamine content of humc over hrs from a mean of ± pg/cell to ± pg/cell. we next assayed release and found that the addition of ng/ml lps to ng/ml rhscf over hrs decreased β-hex release following fcεri crosslinking from ± % to ± %. following fcεri crosslinking, lps did not alter pgd and ltc release. thus, the addition of lps to humc cultured over hr with ng/ml rhscf reduced % β-hex release, but had no effect on pgd and ltc release. effect of lps on wk humc protease expression and composition in short-term cultures we next examined the effect of lps on wk old humc protease expression and composition over hrs. in the presence of ng/ml rhscf and ng/ml lps, facs analysis showed significant increases in tryptase+, chymase+, carboxypeptidase+, tryptase+/chymase+ and tryptase+/carboxypeptidase+ humcs compared to cultures with ng/ml rhscf only. this effect was not observed when cells were cultured in ng/ml of rhscf. as shown in figure 3b, live gate analysis of the cd117low and cd117high humc subpopulations, cultured in the presence of ng/ml rhscf with and without ng/ml lps, determined that increases in chymase and carboxypeptidase staining were detected predominately for the cd117low subpopulation. effect of lps and pgn on wk old humc cytokine release and the effect of cytokines on protease expression since mature wk humc cultures do not contain other cell types, we next asked whether the effect of lps on protease expression might be induced through endogenous humc cytokine release. to this end, cell culture supernatants harvested from humc cultures following hr incubation with or ng/ml rhscf alone, or with ng/ml lps were assayed for the presence of il-1β, il- il- il- il- il- il- ifn-γ, scf and tnf-α. as shown in figure 4a, ng/ml lps induced humc to produce il-1β, il- il- and il- with no differences noted using or ng/ml rhscf. no elevations in il- il- il- ifn-γ and tnf-α were noted in the presence of lps. as expected, scf was detected in cultures containing scf and lps, but levels were not elevated above those measured in cultures with scf alone. in addition, no elevations in cytokines were noted in cultures to which pgn had been added. to determine if observed increases in tryptase, chymase and carboxypeptidase expression could be explained on the basis of endogenous il-1β, il- il- and il- release, humc were cultured with rhscf or ng/ml alone or with rhil-1β, rhil- rhil- and rhil- individually or as a four-cytokine mixture, using concentrations of cytokines detected by immunoassay in the culture supernatants. the combination of ng/ml rhscf with either il-1β or il- gave rise to significant increases in tryptase+, chymase+, tryptase+/chymase+ and tryptase+/carboxypeptidase+ humcs, in contrast to rhil- or rhil- which did not increase tryptase, chymase and carboxypeptidase expression. no increases in protease expression were observed in the presence of ng/ml rhscf with other cytokines. a combination of ng/ml rhscf with pg/ml rhil-1β, pg/ml rhil- pg/ml rhil- and pg/ml rhil- cytokine concentrations detected in the cell cultures supernatant following hr of incubation with ng/ml lps, gave rise to increased tryptase+, chymase+, carboxypeptidase+, tryptase+/chymase+ and tryptase+/carboxypeptidase+ humc numbers, reproducing the observed increases seen with ng/ml lps and ng/ml scf. furthermore, this effect of lps could be significantly reduced when humc were cultured for hr in the added presence of μg/ml anti-il-1β and μg/ml anti-il- antibodies. qpcr analysis of humc cells showed an overall 8-fold increase in tryptase and 20-fold increase in chymase expression, following stimulation for hr with ng/ml rhscf, pg/ml rhil-1β, pg/ml rhil- pg/ml rhil- and pg/ml rhil-. similar increases in tryptase and chymase expression were noted following stimulation for hr with ng/ml rhscf and ng/ml lps. together, this data is consistent with the conclusion that lps stimulates humc in culture to release il-1β, il- il- and il- and the overall net effect is to increase cd117low humc production of tryptase, chymase and carboxypeptidase. effect of lps on 4– wk old wt and tlr ko bmmc protease mrna expression in short-term culture since short-term exposure to lps increased protease expression in humc, we asked whether short-term exposure to lps could also effect the protease mrna expression of bmmc from wt mice. thus, bmmc were first allowed to mature over 4– wks, washed and cultured in either or ng/ml rmil- or ng/ml rmscf alone and with ng/ml lps for hr. bmmc were then harvested and examined by qpcr for mmcp mrna expression. wt bmmc had constitutive mrna expression for the proteases mmcp- - - and cpa when cultured in either rmil- or rmscf for hr. as shown in figure following the addition of lps for hr, an upregulation in mmcp- mmcp- and mmcp- mrna expression was observed in the presence of either rmil or in the presence of rmscf. a similar pattern of mmcp upregulation was also observed at the higher concentrations of rmil or rmscf in the presence of lps. since tlr deficient mice are hyporesponsive to lipopolysaccharide, and to determine if upregulated mmcp levels by lps were mediated by tlr we obtained bmmc from wt c3h/heouj and tlr ko c3h/hej mice and exposed them to low concentrations of cytokine in combination with lps. as can be seen, in the presence of either rmil- or rmscf, wt bmmc from c3h/heouj mice had a similar mmcp mrna profile in response to lps as wt bmmc from c57bl/ mice. however, in contrast with c3h/heouj, bmmc cultured from c3h/hej mice and incubated with mil-, or rmscf had reduced mmcp- mmcp- and mmcp- mrna expression following incubation with lps. bmmc from both wt and tlr ko mice were also cultured in the presence of rmil- or rmscf with pgn or lta for hr, and examined for any effect on protease mrna expression. as shown in figures 5e–h, neither tlr ligand affected mmcp expression in wt or tlr ko mice. taken together these data suggest that lps, but not the tlr ligands pgn or lta, induce the upregulation of mmcp- - and - mrna expression in bmmc through cytokines which are produced by mast cell activation through tlr discussion in this report, we demonstrate that lps and pgn have very different and specific effects on progenitor and mature humc. these observations are relevant, since humc maturation may occur in proximity to tissues such as the lung and gastrointestinal mucosa that interface with the external environment, where humc progenitors and their mature counterparts may variably be exposed to bacterial-derived products over time. it has been shown, for example, that inhalation of aerosolized lps exacerbates allergic airway inflammation in the mouse experimental asthma model through tlr4-mediated mast cell activation and promotion of th responses. cell transfer experiments using tlr ko mast cells confirmed that the effect of lps is mediated through tlr on mast cells, consistent with our data. we also found that humcs in the presence of lps synthesized de novo il-1β and il- in addition to il- and il- which is consistent with the induction of th effector responses reported for mouse dendritic cell cultures. if the hypothesis put forward that, under experimental conditions, toll-mediated events facilitate the generation of th responses, then mouse and human in vitro mast cell data are in agreement, and in vivo inhalation of aerosolized lps might induce both humc and dc to produce increased cytokine levels that lead to the promotion of th1-skewed responses. the net effect of these th responses in combination with lps-induced th responses would determine the overall extent of allergic airway inflammation. we thus examined cd34+-derived humc progenitors over wks and wk mature humc over hr for the effect of lps, pgn and lta on cell numbers; and cd fcεri, cytokine, tryptase, chymase and carboxypeptidase expression. in continuous culture over wks, lps had little or no effect on humc numbers, while pgn led to a decrease in humc numbers at all concentrations. lta, another tlr agonist, also decreased humc numbers at the highest concentrations. lps, but not pgn, led to small increases in cd tryptase and chymase expression compared with rhscf alone, while decreasing fcεri expression. these differential effects of lps and pgn became more evident when mature wk humc were used in short-term cultures. when wk humc, originating from cd34+ cells in rhscf, were cultured with pgn and ng/ml rhscf for hr, pgn reduced humc numbers below those observed with ng/ml rhscf alone. pgn had no effect on humc numbers when ng/ml rhscf was used. lps somewhat enhanced humc numbers in the presence of ng/ml rhscf. fcεri expression was again reduced in cultures containing either or ng/ml rhscf and lps, consistent with the observation that the lta, following ligation with tlr reduced surface expression of fcεri in isolated human pulmonary mast cells. in the presence of ng/ml rhscf, the reduction in fcεri expression could be seen to affect two cd subpopulations: cd117low and cd117high humcs. the mfi of the cd117high cells increased a log-fold higher, suggesting that ng/ml of rhscf suppressed cd expression on humc, consistent with other reports. in the presence of lps and ng/ml, but not ng/ml of rhscf, tryptase positive humc co-expressing chymase and carboxypeptidase increased, and live gate analysis showed that increases in tryptase, chymase and carboxypeptidase occurred predominately in the cd117low subpopulation. these data therefore demonstrate that short-term exposure of mature humc to varying concentrations of rhscf alters cd expression, and protease expression; and that these effects are modulated by lps. of note is that ng/ml rhscf that we and others use for optimal humc in vitro growth may mask the upregulatory effects of lps, when lps is added to cultures. it has been reported that activation of murine mast and dendritic cells through tlrs augments production of inflammatory cytokines, and that cytokines can influence human mast cell protease expression. to determine whether lps-driven short-term cultures induced humc-derived cytokine production which in turn could influence cell survival as well as changes in surface receptor and protease expression, we assayed short-term cultures and found, as noted above, significant levels of il-1β, il- il- and il- in lps-driven, but not pgn-driven, cultures. furthermore, we were able to reproduce the effects of lps on humc by culturing these cells in ng/ml rhscf and immunoassay-detected concentrations of il-1β, il- il- and il-. the combination of ng/ml rhscf with either il-1β or il- but not il- or il- also reproduced the lps observed effects in short-term cultures. thus, one possible mechanism of action of lps is to stimulate production of mature humc-derived il-1β and il- which together promote humc survival, alter humc fcεri and protease expression, and together with il- direct humc involvement in resolving innate immunity and promoting acquired immune responses. this transition from innate to acquired immunity involves the resolution of an inflammatory event, and as demonstrated in mouse models, il- is involved in reducing bacteria-induced inflammation. in addition, although il- was not shown to affect humc survival, surface receptor and protease expression, il- production by humc might in part explain how lps can promote neutrophil influx into inflammatory sites and promote host defense mechanisms. the conclusion that lps upregulates humc protease expression and composition were further supported using bmmc from wt and tlr ko mice. it is known that exposure of wt bmmc to rmil and rmscf alters the protease composition of these cells. following exposure of wt bmmc to reduced concentrations of rmil- or rmscf in the presence of lps an increase in mrna for the mmcp- - and - was observed. bmmc from tlr ko mice, following stimulation with lps, failed to show any alteration in mmcp- mmcp- and mmcp- mrna expression compared with wt controls. these findings demonstrate that the differential mrna expression of mmcp depends on the presence and concentration of rmil- or rmscf used to culture bmmc, and that the mmcp mrna expression of bmmc can be influenced by lps ligation with tlr an effect which is eliminated in the absence of a functional tlr receptor. mmcp- mrna expression in mucosal mast cells of trichinella spiralis-infected mice can similarly be influenced by il-. furthermore, mmcp- mmcp- and mmcp- are chymases, and mmcp- most probably represents the murine homolog of human chymase. in addition, the observation that tlr ligands pgn and lta had no effect on mmcp mrna expression from wt and tlr ko-derived bmmc appears to be consistent with the observation in humc that lps, but not pgn, increases humc numbers and protease expression, and may be related to the lps-induced increase in il-1β and il- the upregulation of human chymase and carboxypeptidase by lps may be another means by which mast cells participate in endotoxin-driven alteration in th and th responses. mast cell-derived chymase and carboxypeptidase are involved in resistance to toxic endogenous or exogenous peptides such as endothelin- and venoms respectively; thus, their induction may counterbalance the effect of tryptase which is known to increase t cell-derived il- and airway hyper-responsiveness.  taken together, these data demonstrate that lps can alter humc numbers, influence cytokine and protease expression and composition, and that the effect of lps is best observed when reduced concentrations of rhscf are used, as may occur in tissues. these data further support the idea that the appearance of lps during a gram negative bacterial infection, in the presence of tissue concentrations of scf, may induce tissue mast cells to express a unique composition of proteases beneficial for controlling and eliminating this particular infection. in other infections or clinical states having higher concentrations of other cytokines and/or the presence of other mediators, tissue mast cell progenitors may differentially develop and express specific surface receptors and proteases which enable these mast cells to more effectively participate in innate immune responses. 
over wks of culture, lps had minimal effect on humc numbers but increased cd tryptase and chymase expression. lps decreased fcεri expression and β-hexosaminidase release; but had no effect on ltc and pgd production. pgn inhibits humc growth, while lps exerts its primary effects on mature humc by altering cytokine production and protease composition, particularly at low concentrations of scf. it is unknown, however, whether long- or short-term exposure to bacteria-derived toll-like receptor ligands, such as lipopolysaccharide or peptidoglycan, influences humc biology. pgn reduced humc numbers; and cd and tryptase expression. human mast cell maturation occurs in tissues interfacing with the external environment, exposing both mast cell progenitors and mature mast cells, to bacteria and their products. for mature mast cells, lps in the presence of rhscf increased cd tryptase, chymase and carboxypeptidase expression, primarily in cd117low humc. these data demonstrate the ability of bacterial products to alter humc mediator production, granular content, and number which may be particularly relevant at mucosal sites where humc are exposed to these products. comparative studies with mouse bone marrow-derived mast cells from wild type, but not tlr knockout mice, showed increases in mrna of mouse mast cell chymases mmcp- mmcp- and mmcp- il-1β and il- were detected in short-term culture supernatants of lps treated cells, and reproduced the increases in cd tryptase, chymase, and carboxypeptidase expression observed in the presence of lps.
 human mast cell maturation occurs in tissues interfacing with the external environment, exposing both mast cell progenitors and mature mast cells, to bacteria and their products. it is unknown, however, whether long- or short-term exposure to bacteria-derived toll-like receptor ligands, such as lipopolysaccharide or peptidoglycan, influences humc biology.  over wks of culture, lps had minimal effect on humc numbers but increased cd tryptase and chymase expression. pgn inhibited humc development. for mature mast cells, lps in the presence of rhscf increased cd tryptase, chymase and carboxypeptidase expression, primarily in cd117low humc. lps decreased fcεri expression and β-hexosaminidase release; but had no effect on ltc and pgd production. pgn reduced humc numbers; and cd and tryptase expression. il-1β and il- were detected in short-term culture supernatants of lps treated cells, and reproduced the increases in cd tryptase, chymase, and carboxypeptidase expression observed in the presence of lps. comparative studies with mouse bone marrow-derived mast cells from wild type, but not tlr knockout mice, showed increases in mrna of mouse mast cell chymases mmcp- mmcp- and mmcp-  pgn inhibits humc growth, while lps exerts its primary effects on mature humc by altering cytokine production and protease composition, particularly at low concentrations of scf. these data demonstrate the ability of bacterial products to alter humc mediator production, granular content, and number which may be particularly relevant at mucosal sites where humc are exposed to these products. 