BACKGROUND
the secretory leukocyte protease inhibitor  is an  <dig>  kda protein originally identified in bodily secretions such as saliva, seminal fluid, and in the mucus of cervical, nasal and bronchial passages  <cit> .
it was later found in neutrophils, peritoneal macrophages, astrocytes and neurons  <cit>  as well as in activated regulatory t cells  <cit> , and was shown to be strongly upregulated in the cns as a consequence of ischemic stroke  <cit> , spinal cord injury  <cit>  and experimental autoimmune encephalomyelitis   <cit> .
slpi is a potent inhibitor of leukocyte serine proteases, including elastase and cathepsin g from neutrophils, chymase and tryptase from mast cells, and trypsin and chymotrypsin from pancreatic acinar cells  <cit> .
in addition, slpi suppresses bacterial growth  <cit> , inhibits hiv- <dig> infection of macrophages  <cit>  and exerts anti-inflammatory functions in macrophages, neutrophils and b cells by inhibition of iκbα degradation  <cit> .
finally, slpi diminishes inflammatory gene expression and inflammatory cell accumulation after hepatic and lung injuries  <cit> , is neuroprotective in an ischemic stroke model  <cit>  and suppresses the expression of matrix metalloproteinases by stimulated monocytes  <cit> .
mice deficient in slpi show impaired cutaneous wound healing with increased inflammation.
additionally, an increased tgf-β activity was found in these mice, likely due to an increased proteolytic activation of latent tgf-β in slpi-deficient animals  <cit> .
slpi-mediated suppression of tgf-β expression by human endometrial cells  <cit>  and slpi's inhibition on the induction of regulatory t cell differentiation by elastase  <cit>  provide corroborating evidence that it has prominent proinflammatory properties.
we investigated whether the effects of slpi on the immune system may have implications in diseases characterized pathologically by inflammation as a result of autoimmune mechanisms such as multiple sclerosis .
indeed slpi is known to be markedly upregulated in a rat model of the disease called experimental autoimmune encephalomyelitis  <cit> .
eae can be induced by immunization with myelin proteins which result in auto-reactive cd4+ t cells to react with myelin and cause concomitant clinical disease.
the inflammatory lesions in eae strongly mimic the acute lesion in ms  <cit> .
based on all these findings our studies aimed to determine the role of slpi in the pathogenesis of eae in sjl/j mice and da rats and to study the impact of slpi on tgf-β activity.
methods
animals
female dark agouti  rats, 6-8-weeks old were purchased from harlan laboratories , and female sjl/j mice, 6-8-weeks old, were purchased from the jackson laboratory .
animals were housed in the animal facility of roosevelt hospital  and were 8-10-week old when used for experiments.
all procedures were conducted according to protocols approved by the iacuc committee of roosevelt hospital.
induction and clinical evaluation of eae
for active eae induction, sjl/j mice were immunized with  <dig> μl of a suspension containing  <dig> μg of murine plp peptide , pepceuticals, leicestershire, uk), and an equal volume of cfa supplemented with  <dig> μg h37ra by subcutaneous injection to generate plp-specific encephalitogenic lymph node cells.
in order to induce the adoptive transfer eae  in sjl/j mice, lymph node cells were harvested  <dig> days after plp-immunization and restimulated in vitro for four days with  <dig> μg/ml plp-peptide.
naive female sjl/j mice were injected intraperitoneally  with  <dig>  ×  <dig> preactivated plp-specific lnc for at-eae induction.
to induce active eae in da rats, animals were immunized subcutaneously at the base of the tail with  <dig> μg mog1- <dig> emulsified in complete freund's adjuvant  supplemented with  <dig> μg of heat-inactivated mycobacterium tuberculosis   in a total volume of  <dig> μl.
animal weight and clinical score were recorded daily .
the mean cumulative score for a treatment group was calculated as the sum of the daily scores of all animals from day zero until the end of the experiment divided by the number of animals in the respective group.
protein vaccination
sjl/j mice and da rats were immunized i.p.
with  <dig> μl of a solution containing  <dig> μg of slpi , respectively, mixed with  <dig> μl of the adjuvant aluminum hydroxide .
vaccinations were performed twice with an interval of  <dig> weeks as previously described  <cit> .
control animals were injected with ova peptide ) and aluminum hydroxide.
after the second vaccination, animals rested  <dig> to  <dig> weeks before eae was induced by adoptive transfer of encephalitogenic lymph node cells  or mog-protein immunization, respectively.
enzyme-linked immunosorbent assay  for anti-slpi antibody detection
a direct elisa assay was used to confirm slpi-specific igg titers in sera from mice that had received the second vaccination two weeks before.
96-well elisa plates  were coated overnight at 4°c with recombinant slpi protein ) at concentrations of  <dig> ng/well.
plates were washed with  <dig> % pbs/tween- <dig>  sera from slpi protein-vaccinated or ova-vaccinated mice or rats were then added in serial dilutions from 1: <dig> to 1: <dig>  and the plates were incubated for  <dig> h at rt.
after washing, goat anti-mouse igg conjugated to alkaline phosphatase or respectively goat anti-rat igg conjugated to alkaline phosphatase  were used as secondary antibodies with p-nitrophenylphosphate  as a substrate.
confirmation of slpi neutralization by serum slpi antibodies
igg antibodies were purified from mice sera by utilizing the mabtrap™ kit  according to the manufacturer's protocol.
antibody titers to slpi and ova were confirmed by an elisa assay as described above.
u <dig> cells  were cultured at a density of  <dig> ×  <dig> cells/ <dig> μl rpmi medium in a 96-well plate with lps ,  <dig> μg/ml slpi and/or  <dig> μg/ml of purified total igg from mice vaccinated with either slpi or ova-peptide.
after  <dig> h the cells were harvested, and rna was isolated using the rneasy plus mini kit .
the il- <dig> expression was quantified using il- <dig> specific taqman probes  and normalized to respective 18s rrna quantities also determined by using taqman probes .
slpi administration
recombinant slpi  was administered ip to female sjl/j mice for the first sixteen days after disease induction the experimental slpi group of mice was injected with slpi , and the control group of mice received  <dig> μl of  <dig> % saline instead.
all mice received the injection ip three times a day.
quantification of tgf-β in serum samples
a direct elisa assay was used to determine tgf-β levels in serum samples isolated from mice on day  <dig> after eae induction and from da rats at day  <dig> after eae induction using the mouse/rat/porcine/canine tgf-β <dig> immunoassay .
total tgf-β quantities were measured by incubating serum samples with  <dig> m hydrogen chloride which activates latent tgf-β.
activated tgf-β serum samples were analyzed without the addition of hcl.
isolation and cultivation of naive human cd4+ t cells
white blood cells were extracted from peripheral blood using cpt bd vacutainer tubes .
naïve human cd4+ t cells were purified according to the naive cd4+ t cell isolation kit ii instructions .
<dig>  naïve human cd4+ t cells per  <dig>  ml of rpmi were cultivated in u bottom-shaped 96-well plates.
cells were stimulated with αcd3- and αcd28-microbeads  in serum-free "stem line t cell expansion medium" .
determination of tgf-β expression by u <dig> cells
u <dig> cells  were cultured at a density of  <dig> ×  <dig> cells/ <dig> μl rpmi medium in a 96-well plate with or without  <dig> ng/ml slpi .
after  <dig> h the cells were harvested, and rna was isolated using the rneasy plus mini kit .
the tgf-β expression was quantified using tgf-β specific taqman probes  and normalized to respective 18s rrna quantities also determined by using taqman probes .
flow cytometry
flow cytometric analyses were carried out with a facsaria flow cytometer .
after staining the cell surfaces, the cells were washed, fixed and permeabilized with the bd cytofix/cytoperm-kit  and stained for intracellular molecules.
the following antibodies were used for murine cells: pacific blue anti-cd <dig>  , pe anti il-17a  and apc anti-ifn-g   and with alexafluor <dig> anti-foxp <dig>  biolegend, san diego, ca).
human cells were stained with efluor <dig> anti-cd <dig> , pe anti-ifn-g , fitc anti-il- <dig> , pe anti cd <dig>   as well as with alexafluor <dig> anti-foxp <dig>  or alexafluor <dig> isotype antibody .
to detect the proliferation of stimulated naïve human cd4+ t cells, cells were stained with  <dig>  μm cfse using the celltrace™ cfse cell proliferation kit  according to the manufacturer's instructions.
the number of divisions was determined by counting the number of peaks.
for the detection of intracellular il- <dig> and ifn-g in cfse stained cells, alexa fluor <dig> anti-il-17a  and pe-cy <dig> anti-ifn-g  antibodies were used.
additionally, antibodies of the same isotype as the foxp <dig> antibody  were used for staining.
statistical analyses
statistical analyses were performed using sigmastat  <dig>  .
comparisons of groups of normal-distributed data were done by student's t-test or an anova analysis.
for non-normally distributed data from the eae experiments the mann-whitney rank sum test was used for comparison.
error bars represent standard deviations unless otherwise indicated.
RESULTS
vaccination of sjl/j mice with slpi protein induces slpi-specific neutralizing antibodies
previously, we reported that slpi is strongly induced within the spinal cord during the disease course of the mog-induced eae of da rats  <cit> .
to further address this observation, we induced neutralizing slpi antibodies in vivo in order to neutralize slpi and to assess its function during cns inflammation.
in particular, we immunized female sjl/j mice with human recombinant slpi protein and control mice with ovalbumin peptide .
vaccinations were performed twice with an interval of three weeks and resulted in high and reproducible antibody titers for slpi .
to confirm that the induced slpi-specific antibodies were able to neutralize slpi's activity, we determined if they were able to prevent the slpi-mediated inhibition of the lps-induced production of il- <dig> by the human monocytic cell line u <dig>  <cit> .
we found that, serum igg from slpi-vaccinated mice neutralized slpi's inhibitory effect on the lps-induced il- <dig> expression.
in contrast, serum igg isolated from control ova-vaccinated mice had no impact on the inhibition of the lps-mediated induction of il- <dig> by slpi .
these data confirmed that protein vaccination with recombinant slpi leads to the generation of slpi-specific antibodies that neutralize biological effects of soluble slpi in vitro.
slpi blockade is protective in passive eae of female sjl/j mice and in active eae of da rats
to investigate the role of slpi in vivo, we examined the consequences of its neutralization in the passive eae of sjl/j mice.
for the induction of adoptive transfer eae, donor sjl/j mice were immunized with plp peptide.
their lymph node cells  were harvested  <dig> days later and restimulated in vitro for four days with plp.
<dig>  ×  <dig> lnc were subsequently transferred into mice which had been immunized with ova- or slpi-protein two months beforehand.
mice vaccinated with slpi protein developed a significantly less severe eae disease course than the ova-immunized animals  correlating with a significantly delayed disease onset in slpi immunized mice compared to ova-immunized mice.
this finding suggests that the neutralization of slpi by the induction of slpi-specific antibodies had an ameliorative effect on the clinical course of eae.
in order to corroborate slpi's pathogenic role in eae, we administered recombinant slpi to sjl/j mice which have been immunized with plp-peptide for active eae induction.
the slpi group was injected intraperitoneally three times a day for sixteen days with slpi , control mice received  <dig> μl of  <dig> % saline instead.
slpi treated mice developed disease signs earlier than the control mice and had a more severe disease course .
after stopping the administration of slpi, the slpi-treated animals became similar to the control mice.
this might be explained by the limited half-life of slpi in vivo  <cit> .
the observed increase of disease symptoms of slpi-treated mice after day  <dig> could also have been caused by the induction of slpi antibodies, because we found high serum slpi antibody titers in slpi-immunized mice at day  <dig> after eae induction .
slpi has an inhibitory effect on tgf-β in vivo and in vitro
slpi is known to be an inhibitor of the anti-inflammatory molecule tgf-β by two different mechanisms: suppressing the expression of tgf-β  <cit>  and interfering with tgf-β's proteolytic activation  <cit> .
it was also described to interfere with the differentiation of tgf-β producing regulatory t cells induced by neutrophilic elastase  <cit> .
firstly, we incubated u <dig> cells u <dig> for  <dig> h with  <dig> ng/ml of recombinant slpi, a slpi concentration previously determined to have the greatest impact on the differentiation of neural stem cells  <cit> .
interestingly, slpi strongly suppressed the expression of tgf-β quantified by realtime-pcr .
next, we quantified tgf-β in sera obtained from slpi- and ova-immunized mice at the end of the experiment shown in figure 2a and in serum samples isolated from rats at day  <dig> after eae induction.
total tgf-β serum levels were significantly higher in slpi-immunized than in ova-immunized mice .
slpi-immunized rats had significantly increased total and activated tgf-β level compared to ova-immunized rats on day  <dig> after eae induction  suggesting that slpi inhibits tgf-β production in vivo.
slpi inhibits differentiation of regulatory t cells
as tgf-β favors the generation of regulatory t cells, we investigated if slpi interferes directly with the differentiation of regulatory t cells.
naïve human cd4+ t cells isolated from human blood were incubated for six days in serum-free medium in the presence or absence of  <dig> ng/ml slpi protein.
we detected fewer foxp3+ cd25hi cd4+ regulatory t cells in those t cell cultures treated with recombinant slpi protein  indicating that the slpi decreases the generation of regulatory t cells.
by contrast there were no differences in the number of th <dig> and th <dig> cells between slpi treated- and control treated t cell cultures; no th <dig> cells were detectable and less than 1% of the cd4+ t cells were th <dig> cells .
we next determined whether the slpi-mediated decrease of cd4+ foxp3+ t cells correlated with a reduced regulatory activity of slpi-treated naïve human cd4+ t cells.
human naïve cd4+ t cells were stimulated with anti cd3- and cd28-antibody-coated beads with or without  <dig> ng/ml of slpi as previously described.
after four days, cells were further cultured at various ratios with fresh naïve cd4+ t cells isolated from the same donor and stained with cfse.
facs analyses revealed that csfe labeled cd4+ t cells cocultured with slpi treated t cells for four days showed significantly more proliferation than cells cocultured with control treated t cells  confirming that slpi interferes with the regulatory activity of t cell cultures.
to validate that the observed effect on regulatory activity is mediated by suppression of tgf- β, naïve slpi-treated cd4+ t cell cultures were supplemented with active human tgf-β  and incubated for three days.
the addition of only  <dig> pg/ml of tgf- β neutralized the effect of slpi on the differentiation on foxp3+ regulatory t cells completely  corroborating that slpi is interfering with the differentiation of regulatory t cells by inhibiting the activation of tgf- β.
we also addressed the in vivo influence of slpi on regulatory t cell differentiation.
in particular, we examined if the neutralization of slpi decreases the number of these anti-inflammatory t cells.
slpi- and ova-immunized sjl/j mice were sacrificed before eae onset on day  <dig> after induction of adoptive transfer eae and cells were extracted from inguinal lymph nodes.
analogous to the in vitro experiment, mice immunized with slpi-protein showed increased numbers of cd4+ foxp3+ regulatory t cells in lymph nodes compared to ova-immunized animals  providing further evidence that the neutralization of slpi increases the generation of regulatory t cells.
on the other hand, slpi-immunization did not cause any difference in the number of th <dig> and th <dig> cells in lymph nodes at the end of the eae experiment presented in figure 2a.
slpi-and ova-immunized mice were sacrificed on day  <dig> after induction of adoptive transfer eae, their lymph nodes extracted and stimulated in vitro with pma, ionomycin and brefeldin a for intracellular cytokine staining.
no differences in the number of th <dig> and th <dig> cells were found .
discussion
ms is a disease of unknown etiology in which there is substantial clinical and pathological evidence of auto-immune dysfunction  <cit> .
the disease activity in ms at any time point likely reflects the state of equilibrium between pro-inflammatory and anti-inflammatory signals.
systemic immune activation, the integrity of the blood-brain barrier, and inflammatory events within the brain and spinal cord all contribute to demyelination and axonal damage  <cit> .
the role of proteases in ms pathogenesis is of interest because they have crucial regulatory roles in inflammatory processes through a number of different mechanisms.
these include modification and induction of chemokines and cytokines, activation of cellular receptors, cleavage of adhesion molecules, activation of tlr <dig> and matrix metalloproteinases  .
in cns inflammation in particular, mmps may contribute to inflammatory damage through disruption of the blood brain barrier  <cit>  and by cleaving myelin proteins  <cit> .
further investigation and understanding of the role of proteases in ms is therefore important to enable development of therapeutic strategies that modulate cns inflammation  <cit> .
to further elucidate the role of proteases in ms we studied the effects of the protease inhibitor slpi in eae, the accepted disease model of ms. slpi is of interest because in addition to its competitive inhibition of several proteases  <cit> , it is upregulated in eae  <cit>  and has both anti-and pro-inflammatory on the immune system.
slpi exerts anti-inflammatory effects on macrophages, neutrophils  <cit>  and b cells  <cit> .
it also regulates inflammatory responses by reducing inflammatory gene expression and diminishing inflammatory cell accumulation following tissue injury  <cit> .
however, mice deficient in slpi have increased tgf-β activity suggesting a pro-inflammatory slpi function  <cit> .
moreover, slpi suppresses tgf-β  <cit>  and reduces the differentiation of human regulatory t cells induced by elastase  <cit> .
we examined the role of slpi in two different eae models, namely, the plp-induced eae of sjl/j mice and the mog-induced eae in da rats.
while the former is a versatile model of relapsing t-cell mediated cns inflammation  <cit> , the latter, with features such as relapses, chronicity, extensive demyelination and contribution of both cellular and antibody-mediated effects, more closely resembles ms  <cit> .
in order to study the role of slpi within these animal models, we induced slpi-specific antibodies by immunizing animals with recombinant slpi protein together with the adjuvant aluminum hydroxide.
control animals were vaccinated with ovalbumin peptide and adjuvant.
we cannot completely exclude that slpi protein differs in its immunogenicity from ovalbumin peptide resulting in an unspecific modulation of the animals' immune system, but we did not find a difference in the number of th <dig> or th <dig> in the lymph nodes of slpi- and ova-immunized mice during eae.
moreover, we waited six to eight weeks after the second slpi vaccination before inducing eae in order to reduce the nonspecific effects of the immunization procedure on eae pathogenesis.
we tested the antibodies' capacity to neutralize slpi in vitro.
only igg isolated from slpi-immunized but not from control-vaccinated animals prevented slpi's inhibition of lps-induced il- <dig> induction  <cit> .
this data strongly suggests that we were able to induce slpi-specific antibodies which block slpi mediated effects and this effect was not a function of a generalized immune activation.
despite the well-established influence of slpi on immune reactions, this is the first report showing that slpi influences inflammatory cns diseases.
in our experiments, mice which received recombinant slpi for the first sixteen days after eae induction developed a more severe disease course than control injected mice.
after day  <dig> the two treatment groups had a similar disease severity, probably due to the limited half-life of slpi  <cit>  and to the induction of slpi-specific antibodies in vivo.
on the other hand, the neutralization of slpi by generating functional anti-slpi antibodies induced by slpi protein immunization of sjl/j mice and da rats resulted in decreased eae disease severity in both ms models.
we speculate accordingly that the protection mediated by slpi antibodies is a general phenomenon which is independent of the myelin antigen used for eae induction as well as of the procedure to induce eae.
the milder disease course in slpi-immunized rats and mice was paralleled by increased sera levels of tgf-β compared to ova-immunized animals.
these data suggest that in eae, the pro-inflammatory functions of slpi probably associated with down regulation of tgf-β described above have a greater impact than slpi's anti-inflammatory effects.
tgf-β controls proliferation and differentiation of a number of cell types involved in acquired and innate immunity .
in particular reference to ms, tgf-β upregulates cd25+ foxp3+ regulatory t-cells and systemic administration of tgf-β is associated with amelioration of inflammatory activity in eae  <cit> .
in our studies the mechanism of amelioration of disease severity in eae associated with anti-slpi antibodies is not established, but we hypothesized that it is in part due to the increased tgf-β serum levels which may enhance the generation of anti-inflammatory cd25+, foxp3+ regulatory t-cells.
indeed in our eae disease mouse model we found an increased number of cd25hi, foxp3+ regulatory t-cells in lymph nodes compared the numbers seen in control animals.
furthermore, slpi suppresses the expression of tgf-β in monocytic u <dig> cells, and mitogenically stimulated naïve human cd4+ t cell cultures derived from healthy donors contained significantly fewer regulatory t cells when incubated with slpi protein.
additionally, slpi treated t cell cultures showed a decreased ability to regulate the proliferation of cfse labeled cd4+ t cells compared to results with cultures not containing slpi.
supplementation of very small amounts of active tgf-β neutralized slpi's inhibitory impact on the generation of regulatory t cells completely.
the mechanism by which slpi regulates the expression of tgf-β-remains to be elucidated.
slpi's ability to inhibit the activation of nfκb may contribute to tgf-β, as the activation of nfκb induces tgf-β  <cit> .
immature dendritic cells exposed to elastase secrete higher amounts of tgf-β  <cit> .
hence, the inhibition of elastase activity by slpi  <cit>  may also contribute to the observed suppression of tgf-β by slpi in our studies.
these experiments support our hypothesis that the neutralization of slpi is protective in adoptive transfer and active eae probably because of an increase of tgf-β serum level promoting the differentiation of regulatory t cells.
in previous work we had shown increased spinal expression of slpi during mog-induced eae of da rats and had speculated that slpi was protective in the disease model because of its anti-inflammatory functions  <cit> .
our current experiments, however, suggest that the neutralization of slpi effects in eae leads to elevated tgf-β serum levels and is disease protective.
thus, the increased expression of slpi in eae in our previous work is likely a deleterious factor counteracting the tgf-β-mediated regulatory immune responses in the cns.
CONCLUSIONS
the influence of slpi on cns autoimmunity was not addressed before.
despite slpi's manifold anti-inflammatory properties, the severity of both, rat murine eae, was reduced by the induction of neutralizing slpi antibodies.
we provide evidence that slpi exerts pro-inflammatory functions by interference with the production and/or activation of the anti-inflammatory molecule tgf-β.
further studies are warranted to define slpi's role within cns autoimmunity.
competing interests
the authors declare that they have no competing interests.
authors' contributions
amm conceived the design of the study, performed experiments as well as experimental analysis and prepared the manuscript.
ej and hc provided technical support for the experiments and for data collection and drafted the manuscript.
sas provided guidance for all aspects of the work.
all authors read and approved the final version of the manuscript.
supplementary material
additional file 1
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additional file 2
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additional file 3
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additional file 4
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 the secretory leukocyte protease inhibitor is an kda protein originally identified in bodily secretions such as saliva, seminal fluid, and in the mucus of cervical, nasal and bronchial passages. it was later found in neutrophils, peritoneal macrophages, astrocytes and neurons as well as in activated regulatory t cells, and was shown to be strongly upregulated in the cns as a consequence of ischemic stroke, spinal cord injury and experimental autoimmune encephalomyelitis. slpi is a potent inhibitor of leukocyte serine proteases, including elastase and cathepsin g from neutrophils, chymase and tryptase from mast cells, and trypsin and chymotrypsin from pancreatic acinar cells. in addition, slpi suppresses bacterial growth, inhibits hiv- infection of macrophages and exerts anti-inflammatory functions in macrophages, neutrophils and b cells by inhibition of iκbα degradation. finally, slpi diminishes inflammatory gene expression and inflammatory cell accumulation after hepatic and lung injuries, is neuroprotective in an ischemic stroke model and suppresses the expression of matrix metalloproteinases by stimulated monocytes. mice deficient in slpi show impaired cutaneous wound healing with increased inflammation. additionally, an increased tgf-β activity was found in these mice, likely due to an increased proteolytic activation of latent tgf-β in slpi-deficient animals. slpi-mediated suppression of tgf-β expression by human endometrial cells and slpi's inhibition on the induction of regulatory t cell differentiation by elastase provide corroborating evidence that it has prominent proinflammatory properties. we investigated whether the effects of slpi on the immune system may have implications in diseases characterized pathologically by inflammation as a result of autoimmune mechanisms such as multiple sclerosis. indeed slpi is known to be markedly upregulated in a rat model of the disease called experimental autoimmune encephalomyelitis. eae can be induced by immunization with myelin proteins which result in auto-reactive cd4+ t cells to react with myelin and cause concomitant clinical disease. the inflammatory lesions in eae strongly mimic the acute lesion in ms. based on all these findings our studies aimed to determine the role of slpi in the pathogenesis of eae in sjl/j mice and da rats and to study the impact of slpi on tgf-β activity. methods animals female dark agouti rats, 6-8-weeks old were purchased from harlan laboratories, and female sjl/j mice, 6-8-weeks old, were purchased from the jackson laboratory. animals were housed in the animal facility of roosevelt hospital and were 8-10-week old when used for experiments. all procedures were conducted according to protocols approved by the iacuc committee of roosevelt hospital. induction and clinical evaluation of eae for active eae induction, sjl/j mice were immunized with μl of a suspension containing μg of murine plp peptide, pepceuticals, leicestershire, uk), and an equal volume of cfa supplemented with μg h37ra by subcutaneous injection to generate plp-specific encephalitogenic lymph node cells. in order to induce the adoptive transfer eae in sjl/j mice, lymph node cells were harvested days after plp-immunization and restimulated in vitro for four days with μg/ml plp-peptide. naive female sjl/j mice were injected intraperitoneally with × preactivated plp-specific lnc for at-eae induction. to induce active eae in da rats, animals were immunized subcutaneously at the base of the tail with μg mog1- emulsified in complete freund's adjuvant supplemented with μg of heat-inactivated mycobacterium tuberculosis in a total volume of μl. animal weight and clinical score were recorded daily. the mean cumulative score for a treatment group was calculated as the sum of the daily scores of all animals from day zero until the end of the experiment divided by the number of animals in the respective group. protein vaccination sjl/j mice and da rats were immunized i.p. with μl of a solution containing μg of slpi, respectively, mixed with μl of the adjuvant aluminum hydroxide. vaccinations were performed twice with an interval of weeks as previously described. control animals were injected with ova peptide ) and aluminum hydroxide. after the second vaccination, animals rested to weeks before eae was induced by adoptive transfer of encephalitogenic lymph node cells or mog-protein immunization, respectively. enzyme-linked immunosorbent assay for anti-slpi antibody detection a direct elisa assay was used to confirm slpi-specific igg titers in sera from mice that had received the second vaccination two weeks before. 96-well elisa plates were coated overnight at 4°c with recombinant slpi protein ) at concentrations of ng/well. plates were washed with % pbs/tween- sera from slpi protein-vaccinated or ova-vaccinated mice or rats were then added in serial dilutions from 1: to 1: and the plates were incubated for h at rt. after washing, goat anti-mouse igg conjugated to alkaline phosphatase or respectively goat anti-rat igg conjugated to alkaline phosphatase were used as secondary antibodies with p-nitrophenylphosphate as a substrate. confirmation of slpi neutralization by serum slpi antibodies igg antibodies were purified from mice sera by utilizing the mabtrap™ kit according to the manufacturer's protocol. antibody titers to slpi and ova were confirmed by an elisa assay as described above. u cells were cultured at a density of × cells/ μl rpmi medium in a 96-well plate with lps, μg/ml slpi and/or μg/ml of purified total igg from mice vaccinated with either slpi or ova-peptide. after h the cells were harvested, and rna was isolated using the rneasy plus mini kit. the il- expression was quantified using il- specific taqman probes and normalized to respective 18s rrna quantities also determined by using taqman probes. slpi administration recombinant slpi was administered ip to female sjl/j mice for the first sixteen days after disease induction the experimental slpi group of mice was injected with slpi, and the control group of mice received μl of % saline instead. all mice received the injection ip three times a day. quantification of tgf-β in serum samples a direct elisa assay was used to determine tgf-β levels in serum samples isolated from mice on day after eae induction and from da rats at day after eae induction using the mouse/rat/porcine/canine tgf-β immunoassay. total tgf-β quantities were measured by incubating serum samples with m hydrogen chloride which activates latent tgf-β. activated tgf-β serum samples were analyzed without the addition of hcl. isolation and cultivation of naive human cd4+ t cells white blood cells were extracted from peripheral blood using cpt bd vacutainer tubes. naïve human cd4+ t cells were purified according to the naive cd4+ t cell isolation kit ii instructions. naïve human cd4+ t cells per ml of rpmi were cultivated in u bottom-shaped 96-well plates. cells were stimulated with αcd3- and αcd28-microbeads in serum-free "stem line t cell expansion medium". determination of tgf-β expression by u cells u cells were cultured at a density of × cells/ μl rpmi medium in a 96-well plate with or without ng/ml slpi. after h the cells were harvested, and rna was isolated using the rneasy plus mini kit. the tgf-β expression was quantified using tgf-β specific taqman probes and normalized to respective 18s rrna quantities also determined by using taqman probes. flow cytometry flow cytometric analyses were carried out with a facsaria flow cytometer. after staining the cell surfaces, the cells were washed, fixed and permeabilized with the bd cytofix/cytoperm-kit and stained for intracellular molecules. the following antibodies were used for murine cells: pacific blue anti-cd, pe anti il-17a and apc anti-ifn-g and with alexafluor anti-foxp biolegend, san diego, ca). human cells were stained with efluor anti-cd, pe anti-ifn-g, fitc anti-il-, pe anti cd as well as with alexafluor anti-foxp or alexafluor isotype antibody. to detect the proliferation of stimulated naïve human cd4+ t cells, cells were stained with μm cfse using the celltrace™ cfse cell proliferation kit according to the manufacturer's instructions. the number of divisions was determined by counting the number of peaks. for the detection of intracellular il- and ifn-g in cfse stained cells, alexa fluor anti-il-17a and pe-cy anti-ifn-g antibodies were used. additionally, antibodies of the same isotype as the foxp antibody were used for staining. statistical analyses statistical analyses were performed using sigmastat. comparisons of groups of normal-distributed data were done by student's t-test or an anova analysis. for non-normally distributed data from the eae experiments the mann-whitney rank sum test was used for comparison. error bars represent standard deviations unless otherwise indicated.  vaccination of sjl/j mice with slpi protein induces slpi-specific neutralizing antibodies previously, we reported that slpi is strongly induced within the spinal cord during the disease course of the mog-induced eae of da rats. to further address this observation, we induced neutralizing slpi antibodies in vivo in order to neutralize slpi and to assess its function during cns inflammation. in particular, we immunized female sjl/j mice with human recombinant slpi protein and control mice with ovalbumin peptide. vaccinations were performed twice with an interval of three weeks and resulted in high and reproducible antibody titers for slpi. to confirm that the induced slpi-specific antibodies were able to neutralize slpi's activity, we determined if they were able to prevent the slpi-mediated inhibition of the lps-induced production of il- by the human monocytic cell line u. we found that, serum igg from slpi-vaccinated mice neutralized slpi's inhibitory effect on the lps-induced il- expression. in contrast, serum igg isolated from control ova-vaccinated mice had no impact on the inhibition of the lps-mediated induction of il- by slpi. these data confirmed that protein vaccination with recombinant slpi leads to the generation of slpi-specific antibodies that neutralize biological effects of soluble slpi in vitro. slpi blockade is protective in passive eae of female sjl/j mice and in active eae of da rats to investigate the role of slpi in vivo, we examined the consequences of its neutralization in the passive eae of sjl/j mice. for the induction of adoptive transfer eae, donor sjl/j mice were immunized with plp peptide. their lymph node cells were harvested days later and restimulated in vitro for four days with plp. × lnc were subsequently transferred into mice which had been immunized with ova- or slpi-protein two months beforehand. mice vaccinated with slpi protein developed a significantly less severe eae disease course than the ova-immunized animals correlating with a significantly delayed disease onset in slpi immunized mice compared to ova-immunized mice. this finding suggests that the neutralization of slpi by the induction of slpi-specific antibodies had an ameliorative effect on the clinical course of eae. in order to corroborate slpi's pathogenic role in eae, we administered recombinant slpi to sjl/j mice which have been immunized with plp-peptide for active eae induction. the slpi group was injected intraperitoneally three times a day for sixteen days with slpi, control mice received μl of % saline instead. slpi treated mice developed disease signs earlier than the control mice and had a more severe disease course. after stopping the administration of slpi, the slpi-treated animals became similar to the control mice. this might be explained by the limited half-life of slpi in vivo. the observed increase of disease symptoms of slpi-treated mice after day could also have been caused by the induction of slpi antibodies, because we found high serum slpi antibody titers in slpi-immunized mice at day after eae induction. slpi has an inhibitory effect on tgf-β in vivo and in vitro slpi is known to be an inhibitor of the anti-inflammatory molecule tgf-β by two different mechanisms: suppressing the expression of tgf-β and interfering with tgf-β's proteolytic activation. it was also described to interfere with the differentiation of tgf-β producing regulatory t cells induced by neutrophilic elastase. firstly, we incubated u cells u for h with ng/ml of recombinant slpi, a slpi concentration previously determined to have the greatest impact on the differentiation of neural stem cells. interestingly, slpi strongly suppressed the expression of tgf-β quantified by realtime-pcr. next, we quantified tgf-β in sera obtained from slpi- and ova-immunized mice at the end of the experiment shown in figure 2a and in serum samples isolated from rats at day after eae induction. total tgf-β serum levels were significantly higher in slpi-immunized than in ova-immunized mice. slpi-immunized rats had significantly increased total and activated tgf-β level compared to ova-immunized rats on day after eae induction suggesting that slpi inhibits tgf-β production in vivo. slpi inhibits differentiation of regulatory t cells as tgf-β favors the generation of regulatory t cells, we investigated if slpi interferes directly with the differentiation of regulatory t cells. naïve human cd4+ t cells isolated from human blood were incubated for six days in serum-free medium in the presence or absence of ng/ml slpi protein. we detected fewer foxp3+ cd25hi cd4+ regulatory t cells in those t cell cultures treated with recombinant slpi protein indicating that the slpi decreases the generation of regulatory t cells. by contrast there were no differences in the number of th and th cells between slpi treated- and control treated t cell cultures; no th cells were detectable and less than 1% of the cd4+ t cells were th cells. we next determined whether the slpi-mediated decrease of cd4+ foxp3+ t cells correlated with a reduced regulatory activity of slpi-treated naïve human cd4+ t cells. human naïve cd4+ t cells were stimulated with anti cd3- and cd28-antibody-coated beads with or without ng/ml of slpi as previously described. after four days, cells were further cultured at various ratios with fresh naïve cd4+ t cells isolated from the same donor and stained with cfse. facs analyses revealed that csfe labeled cd4+ t cells cocultured with slpi treated t cells for four days showed significantly more proliferation than cells cocultured with control treated t cells confirming that slpi interferes with the regulatory activity of t cell cultures. to validate that the observed effect on regulatory activity is mediated by suppression of tgf- β, naïve slpi-treated cd4+ t cell cultures were supplemented with active human tgf-β and incubated for three days. the addition of only pg/ml of tgf- β neutralized the effect of slpi on the differentiation on foxp3+ regulatory t cells completely corroborating that slpi is interfering with the differentiation of regulatory t cells by inhibiting the activation of tgf- β. we also addressed the in vivo influence of slpi on regulatory t cell differentiation. in particular, we examined if the neutralization of slpi decreases the number of these anti-inflammatory t cells. slpi- and ova-immunized sjl/j mice were sacrificed before eae onset on day after induction of adoptive transfer eae and cells were extracted from inguinal lymph nodes. analogous to the in vitro experiment, mice immunized with slpi-protein showed increased numbers of cd4+ foxp3+ regulatory t cells in lymph nodes compared to ova-immunized animals providing further evidence that the neutralization of slpi increases the generation of regulatory t cells. on the other hand, slpi-immunization did not cause any difference in the number of th and th cells in lymph nodes at the end of the eae experiment presented in figure 2a. slpi-and ova-immunized mice were sacrificed on day after induction of adoptive transfer eae, their lymph nodes extracted and stimulated in vitro with pma, ionomycin and brefeldin a for intracellular cytokine staining. no differences in the number of th and th cells were found. discussion ms is a disease of unknown etiology in which there is substantial clinical and pathological evidence of auto-immune dysfunction. the disease activity in ms at any time point likely reflects the state of equilibrium between pro-inflammatory and anti-inflammatory signals. systemic immune activation, the integrity of the blood-brain barrier, and inflammatory events within the brain and spinal cord all contribute to demyelination and axonal damage. the role of proteases in ms pathogenesis is of interest because they have crucial regulatory roles in inflammatory processes through a number of different mechanisms. these include modification and induction of chemokines and cytokines, activation of cellular receptors, cleavage of adhesion molecules, activation of tlr and matrix metalloproteinases. in cns inflammation in particular, mmps may contribute to inflammatory damage through disruption of the blood brain barrier and by cleaving myelin proteins. further investigation and understanding of the role of proteases in ms is therefore important to enable development of therapeutic strategies that modulate cns inflammation. to further elucidate the role of proteases in ms we studied the effects of the protease inhibitor slpi in eae, the accepted disease model of ms. slpi is of interest because in addition to its competitive inhibition of several proteases, it is upregulated in eae and has both anti-and pro-inflammatory on the immune system. slpi exerts anti-inflammatory effects on macrophages, neutrophils and b cells. it also regulates inflammatory responses by reducing inflammatory gene expression and diminishing inflammatory cell accumulation following tissue injury. however, mice deficient in slpi have increased tgf-β activity suggesting a pro-inflammatory slpi function. moreover, slpi suppresses tgf-β and reduces the differentiation of human regulatory t cells induced by elastase. we examined the role of slpi in two different eae models, namely, the plp-induced eae of sjl/j mice and the mog-induced eae in da rats. while the former is a versatile model of relapsing t-cell mediated cns inflammation, the latter, with features such as relapses, chronicity, extensive demyelination and contribution of both cellular and antibody-mediated effects, more closely resembles ms. in order to study the role of slpi within these animal models, we induced slpi-specific antibodies by immunizing animals with recombinant slpi protein together with the adjuvant aluminum hydroxide. control animals were vaccinated with ovalbumin peptide and adjuvant. we cannot completely exclude that slpi protein differs in its immunogenicity from ovalbumin peptide resulting in an unspecific modulation of the animals' immune system, but we did not find a difference in the number of th or th in the lymph nodes of slpi- and ova-immunized mice during eae. moreover, we waited six to eight weeks after the second slpi vaccination before inducing eae in order to reduce the nonspecific effects of the immunization procedure on eae pathogenesis. we tested the antibodies' capacity to neutralize slpi in vitro. only igg isolated from slpi-immunized but not from control-vaccinated animals prevented slpi's inhibition of lps-induced il- induction. this data strongly suggests that we were able to induce slpi-specific antibodies which block slpi mediated effects and this effect was not a function of a generalized immune activation. despite the well-established influence of slpi on immune reactions, this is the first report showing that slpi influences inflammatory cns diseases. in our experiments, mice which received recombinant slpi for the first sixteen days after eae induction developed a more severe disease course than control injected mice. after day the two treatment groups had a similar disease severity, probably due to the limited half-life of slpi and to the induction of slpi-specific antibodies in vivo. on the other hand, the neutralization of slpi by generating functional anti-slpi antibodies induced by slpi protein immunization of sjl/j mice and da rats resulted in decreased eae disease severity in both ms models. we speculate accordingly that the protection mediated by slpi antibodies is a general phenomenon which is independent of the myelin antigen used for eae induction as well as of the procedure to induce eae. the milder disease course in slpi-immunized rats and mice was paralleled by increased sera levels of tgf-β compared to ova-immunized animals. these data suggest that in eae, the pro-inflammatory functions of slpi probably associated with down regulation of tgf-β described above have a greater impact than slpi's anti-inflammatory effects. tgf-β controls proliferation and differentiation of a number of cell types involved in acquired and innate immunity. in particular reference to ms, tgf-β upregulates cd25+ foxp3+ regulatory t-cells and systemic administration of tgf-β is associated with amelioration of inflammatory activity in eae. in our studies the mechanism of amelioration of disease severity in eae associated with anti-slpi antibodies is not established, but we hypothesized that it is in part due to the increased tgf-β serum levels which may enhance the generation of anti-inflammatory cd25+, foxp3+ regulatory t-cells. indeed in our eae disease mouse model we found an increased number of cd25hi, foxp3+ regulatory t-cells in lymph nodes compared the numbers seen in control animals. furthermore, slpi suppresses the expression of tgf-β in monocytic u cells, and mitogenically stimulated naïve human cd4+ t cell cultures derived from healthy donors contained significantly fewer regulatory t cells when incubated with slpi protein. additionally, slpi treated t cell cultures showed a decreased ability to regulate the proliferation of cfse labeled cd4+ t cells compared to results with cultures not containing slpi. supplementation of very small amounts of active tgf-β neutralized slpi's inhibitory impact on the generation of regulatory t cells completely. the mechanism by which slpi regulates the expression of tgf-β-remains to be elucidated. slpi's ability to inhibit the activation of nfκb may contribute to tgf-β, as the activation of nfκb induces tgf-β. immature dendritic cells exposed to elastase secrete higher amounts of tgf-β. hence, the inhibition of elastase activity by slpi may also contribute to the observed suppression of tgf-β by slpi in our studies. these experiments support our hypothesis that the neutralization of slpi is protective in adoptive transfer and active eae probably because of an increase of tgf-β serum level promoting the differentiation of regulatory t cells. in previous work we had shown increased spinal expression of slpi during mog-induced eae of da rats and had speculated that slpi was protective in the disease model because of its anti-inflammatory functions. our current experiments, however, suggest that the neutralization of slpi effects in eae leads to elevated tgf-β serum levels and is disease protective. thus, the increased expression of slpi in eae in our previous work is likely a deleterious factor counteracting the tgf-β-mediated regulatory immune responses in the cns.  the influence of slpi on cns autoimmunity was not addressed before. despite slpi's manifold anti-inflammatory properties, the severity of both, rat murine eae, was reduced by the induction of neutralizing slpi antibodies. we provide evidence that slpi exerts pro-inflammatory functions by interference with the production and/or activation of the anti-inflammatory molecule tgf-β. further studies are warranted to define slpi's role within cns autoimmunity. competing interests the authors declare that they have no competing interests. authors' contributions amm conceived the design of the study, performed experiments as well as experimental analysis and prepared the manuscript. ej and hc provided technical support for the experiments and for data collection and drafted the manuscript. sas provided guidance for all aspects of the work. all authors read and approved the final version of the manuscript. supplementary material additional file 1 click here for file additional file 2 click here for file additional file 3 click here for file additional file 4 click here for file
slpi neutralization was associated with increased serum levels of tgf-β and increased numbers of foxp3+ cd4+ t cells in lymph nodes. in vitro, the addition of slpi significantly decreased the number of functional foxp3+ cd25hi cd4+ regulatory t cells in cultures of naive human cd4+ t cells. adding recombinant tgf-β to slpi-treated human t cell cultures neutralized slpi's inhibitory effect on regulatory t cell differentiation. the induction of slpi neutralizing antibodies resulted in a milder disease course in mouse and rat eae. the secretory leukocyte protease inhibitor exerts wide ranging effects on inflammatory pathways and is upregulated in eae but the biological role of slpi in eae, an animal model of multiple sclerosis is unknown
 the secretory leukocyte protease inhibitor exerts wide ranging effects on inflammatory pathways and is upregulated in eae but the biological role of slpi in eae, an animal model of multiple sclerosis is unknown methods to investigate the pathophysiological effects of slpi within eae, we induced slpi-neutralizing antibodies in mice and rats to determine the clinical severity of the disease. in addition we studied the effects of slpi on the anti-inflammatory cytokine tgf-β.  the induction of slpi neutralizing antibodies resulted in a milder disease course in mouse and rat eae. slpi neutralization was associated with increased serum levels of tgf-β and increased numbers of foxp3+ cd4+ t cells in lymph nodes. in vitro, the addition of slpi significantly decreased the number of functional foxp3+ cd25hi cd4+ regulatory t cells in cultures of naive human cd4+ t cells. adding recombinant tgf-β to slpi-treated human t cell cultures neutralized slpi's inhibitory effect on regulatory t cell differentiation.  in eae, slpi exerts potent pro-inflammatory actions by modulation of t-cell activity and its neutralization may be beneficial for the disease. slpieaetgf-betamultiple sclerosis 