a cornerstone of the inflammatory response is the highly coordinated interplay of inflammatory regulators and immune cell migration to sites of injury or infection [ 13 ] . immune cells act in concert to mitigate adverse responses resulting from exposure to toxins , bacteria , viruses , or various pathogens . regulation of innate and adaptive immune responses is a tightly controlled process involving a plethora of different cell types that interact with and respond to changing levels of immune mediators . migration and accumulation of immune cells at sites of injury or infection eventually result in the resolution of inflammation and repair of damaged tissue . however , dysregulation of immune surveillance and activation of immune responses toward self - antigens can lead to adverse effects including autoimmune diseases such as rheumatoid arthritis , inflammatory bowel disease , and relapsing - remitting multiple sclerosis ( ms ) . cytokines and chemokines are members of two different families of immune mediators that each play an indispensable role in the control and regulation of an immune response . they differ based on their chromosome location , cell specificity , protein structure and function , cell surface receptor , and cellular signaling pathways . the immune system makes use of these immune mediators to coordinate and efficiently direct immune responses [ 8 , 9 ] . interferons ( ifns ) are part of the cytokine family of proteins and are comprised of two different groups : type i ( ifn- , - ) and type ii ( ifn- ) . type i and type ii ifns differ in structure , cell surface receptor , and , to some extent , cellular signaling . type ii ifns have been associated with proinflammatory effects in contrast to type i ifns which appear anti - inflammatory . ifn- and ifn- are located on different chromosomes and exhibit a high degree of homology at both the amino acid and nucleotide levels . type i ifns bind to the same heterodimeric receptor although receptor assembly and signaling events can differ [ 10 , 11 ] . activation of the ifn receptor leads to a cascade of intracellular signaling events involving the jak - stat signaling pathway . activation of the ifn - receptor has been demonstrated to result in the differential induction of genes that protect against viral infection , regulate immune cell activity , control cell growth , and reduce oxidative stress [ 9 , 12 , 13 ] . ifns are an accepted treatment for hepatitis b , hairy cell leukemia , and malignant melanoma . in addition , betaseron ( ifn-1b ; bayer healthcare ) , a type i interferon , provided the first approved treatment for ms . chemokines are potent immune modulators that act through a specific interaction with g - protein coupled receptors ( gpcr ) . chemokine binding to gpcrs results in important biologic effects [ 15 , 16 ] . like ifns , chemokines and their receptors play a direct role in the regulation of immune responses and are proven drug targets . as much as 2% of the entire human genome encodes genes related to gpcr functions , and close to 50% of all current drugs are modulators of gpcr activity [ 17 , 18 ] . one important aspect of chemokines and their receptors is that they play a central role in the regulation of immune cell migration to sites of inflammation . chemokine - dependent activation of gpcrs causes the activation of heterotrimeric g - protein subunits , leading to regulation of enzymes and ion channels that results in enhanced cell migration and adhesion [ 1821 ] . a recently discovered protein family of negative regulators of g - protein signaling ( rgs ) controls , in part , the extent of g - protein subunit activation . rgs proteins terminate g - protein signaling by accelerating gtp hydrolysis of activated g - protein subunits [ 20 , 21 ] . upregulation of certain rgs proteins has been shown to decrease immune cell migration and reduce chemokine - dependent calcium flux , an established measurement of gpcr signaling . rgs activity appears to be tightly controlled and highly dependent on tissue distribution and expression . although the coordinated response of cytokines and chemokines is known to be important , studies focused on identifying mechanisms by which these two families of immune modulators can work together to elicit an appropriate immune response are limited . in this paper , we describe for the first time the induction of a negative regulator of g - protein signaling 1 ( rgs1 ) by ifn- , suggestive of a functional link between ifn and chemokine signaling . peripheral blood mononuclear cells ( pbmcs ) were obtained from healthy individuals and verified to be free of viral infection ( all cells llc , berkeley , ca ) . pbmcs ( 1  10  cells / ml ) were cultured ( 37c , 5.0% carbon dioxide ( co2 ) ) and grown in eagle 's minimum essential medium supplemented with 10% v / v heat - inactivated fetal bovine serum and 2.0  mm l - glutamine . cells were incubated with or without ifn--1b ( 1000 international units ( iu)/ml , 1  10 cells ) , ifn- ( r&d systems , minneapolis , mn ; 1000  iu / ml , 1  10 cells ) or phorbol myristate acetate ( pma ) ( roche diagnostics , indianapolis , in ; 2.0  ng / ml , 1  10 cells ) for the times indicated . cells were collected , washed twice in cold phosphate - buffered saline ( pbs ) , and rna was isolated . to determine the specificity of induction of rgs1 by ifn- , pbmcs were stimulated with or without ifn- ( 1000  iu / ml , 1  10 cells ) for either 4 or 18 hours . rna isolated at each time point was analyzed by reverse transcription polymerase chain reaction ( rt - pcr ; taqman ) using specific rgs primer sets ( rgs1 , hs00175260_m1 ; rgs2 , hs00263639_m1 ; rgs5 , hs00186212_m1 ; rgs7 , hs00243156_m1 ; rgs12 , hs01017940_m1 ; rgs14 , hs00197761_m1 ) ( applied biosystems , foster city , ca ) . primary cells including b cells , monocytes , and t cells were isolated by negative selection using macs cell separation ( miltenyi biotec inc . purity of each cell population was determined to be greater that 90% by measurement of cell - type - specific surface markers using fluorescent - activated cell sorting ( facs ) ( data not shown ) . isolated cell populations ( 1  10  cells / ml ) were incubated with or without ifn- ( 1000  iu / ml ) for 4 hours . after stimulation , cells were washed in cold pbs , rna isolated , and rgs1 expression determined by taqman . the concentration of antisera needed to neutralize the ifn receptor was determined using a previously characterized ifn - dependent reporter assay . generation of a rabbit polyclonal antisera recognizing the ectodomain of the ifn receptor ( ifnar2c ) has been previously described . a stable cell line ( ht1080luc ) containing an ifn reporter was preincubated with increasing concentrations of either preimmune or ifnar2c - neutralizing antisera for 1 hour at room temperature . cells were then stimulated , with or without ifn- , for 4 hours , and reporter activity was measured after cell lysis using a luciferase substrate ( luciferase assay system , promega , madison , wi ) . the amount of ifnar2c antisera needed to inhibit activation of the ifn - receptor was determined relative to preimmune sera . pbmcs ( 1  10  cell / ml ) were incubated for 1 hour at room temparature in cell - culture media containing preimmune sera or ifnar2c antisera at a concentration able to neutralize ifn - receptor activity by > 95% . cells were stimulated with or without ifn- ( 1000  iu / ml ) for 4 hours followed by cell lysis and rna isolation as previously described . ifn--dependent expression of rgs1 in pbmcs treated with preimmune sera , ifnar2c - neutralizing sera or control ( unstimulated ) samples was determined by taqman as previously described . ms patients were diagnosed with definite ms according to the mcdonald criteria and recruited for this study as previously described [ 13 , 23 ] . patients had an expanded disability status scale score of 2.5  1.2 ; and all patients exhibited characteristic magnetic resonance imaging ( mri ) lesions and two or more episodes of clinically evident relapses and remissions . patients were continually monitored during the course of the study and were free of exacerbations for at least 3 months before and after blood collection . study approval and written consent information were acquired and documented as described previously . the patient cohort used in this study was held nave to immunosuppressants or corticosteroids treatment for 1 year prior to , and during , the study . ms patients were under treatment with betaseron ( ifn- ) following an established clinical protocol for an average of 1.8  0.8 years before beginning the study . after 64 hours a baseline sample ( t = 0 ) was drawn , then a standard dose of ifn- ( 8 million iu , specific activity 2.3  10 iu / mg ) was self - administered subcutaneously under physician supervision , and heparinized blood samples drawn at 4 , 18 , and 42 hours post - ifn- administration . pbmcs were isolated using ficoll - paque plus ( amersham biosciences , nj ) , and purified rna characterized using an agilent 2100 bioanalyzer ( agilent instruments , inc . rna was profiled using affymetrix oligonucleotide microarrays ( affymetrix genechip hg - u133a ; affymetrix inc . , gene expression values were determined using bioconductor 's implementation of the robust multichip average ( rma ) algorithm with default options [ 13 , 24 , 25 ] . gene expression resulting from affymetrix probe - set measurements are presented as rma computations performed with quantile normalization , including standardization and background adjustment [ 13 , 25 ] . genechip probe sets are classified as 25-mer oligonucleotide sequences ( n ~ 11 ) that hybridize to the corresponding nucleotide sequence present in a given gene . gene expression analysis was also performed with the statistical analysis and critical rationalization of expression data ( sacred ) software analysis package developed at berlex biosciences , inc . general methods for performing quantitative rt - pcr ( qrt - pcr ) have been previously described . briefly , expression of rgs1 was measured using rgs1-specific primer sets ( hs00175260_m1 ) ( taqman gene expression assays , applied biosystems , foster city , ca ) . rna samples were isolated using rneasy midi endotoxin - free kit ( qiagen , inc . , santa clara , ca ) and run in triplicate or quadruplicate for each sample . analyzed rna samples were identical to those used for gene chip analysis . pcr reactions were performed using a 96-well opticon ii dna engine ( biorad , hercules , ca ) or an abi prism 7900ht sequence detection system ( applied biosystems , foster city , ca , ) . results are presented as linearized c(t ) values normalized to glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) . rgs1 polyclonal antisera used for immunoprecipation was generated against the 36 n - terminal amino acids of rgs1 ( harlan laboratories , san francisco , ca ) . the amino acid sequence mffsanpkelkgtthsllddkmqkrrrpktfgmdmka was coupled to klh and used to produce rgs1 antisera in rabbits . the immunizing rgs1 peptide sequence was used as an input sequence to query numerous databases using the ncbi basic local alignment search tool ( blast ) for detecting overlapping sequence alignments . using this approach , the immunizing peptide sequence was shown to be specific for rgs1 or rgs1 isoforms with only a minor overlap with a predicted sequence for a g - protein 3-like molecule in oryctolagus cuniculus ( european rabbit ) . rgs1 antisera was first analyzed by elisa to demonstrate immune reactivity against the immunizing peptide coupled to bovine serum albumin . evaluation of rgs1 antiserum was further appraised by immunoblotting using purified rgs1 containing a 6xhistidine tag at the c - terminus . 6xhis rgs1 ( 5  g/200  l sample buffer ) was subjected to sds - page using a large single well comb . following sds - page , proteins were transferred to a polyvinylidene difluoride membrane ( pvdf ) ( pro - blot , appled biosystems , inc . ) and incubated in blocking buffer ( 20  mm tris - hcl ( ph 7.3 ) , 150  mm nacl containing 0.1% tween 20 ) overnight at room temperature . the resultant membrane was then cut into individual strips , and each strip was placed in its own well using an 8 well plastic tray . membrane strips were probed with either preimmune sera , rgs1 antisera , or with a specific antibody recognizing 6xhis ( 6xhis tag antibody - hrp , ab1187 ) ( abcam , cambridge , ma ) . strips probed with the 6xhistag antibody - hrp ( 1  :  1000 dilution ) or rgs1 antisera ( 1  :  1000 dilution ) or preimmune sera ( 1  :  1000 dilution ) were incubated for two hours at room temperature and washed . after washing the membrane strips in blocking buffer , the strips incubated with rgs1 or preimmune antisera were further incubated with a second antibody ( goat anti - rabbit igg - hrp ; 1  :  2000 dilution ) ( abcam , cambridge , ma ) for 1 hour at room temperature followed by washing in blocking buffer . all membrane strips were arranged next to each other on a plastic sheet , and rgs1 ( or 6xhistagged rgs1 ) was detected using a chemiluminescent detection method ( supersignal west pico chemiluminescent kit , pierce / thermo scientific , rockford , il ) . for immunoprecipation of rgs1 , pbmcs were incubated with or without ifn- ( 1000  iu / ml ) , ifn-   ( 1000  iu / ml ) , or pma ( 2  ng / ml ) for 20 hours . an equal number of cells ( 2  10 ) were collected by centrifugation ( 2000    g , 10 minutes ) , washed twice in cold pbs , and lysed in solubilization buffer ( 20  mm tris - hcl ( ph 8.0 ) buffer containing 150  mm sodium chloride ( nacl ) , 1.0% nonidet p-40 ( v / v ) , and 2  mm edta ) at 4c . insoluble material was removed by centrifugation ( 10 minutes , 10  000    g , 4c ) and rgs1 antiserum ( 1  :  500 ) ( described above ) or preimmune ( pi ) ( 1  :  500 ) sera added to each lysate . lysates were incubated overnight at 4c , mixed with protein - g agarose ( boehringer ingelheim , inc . ) , incubated for 1 hour , washed in blocking buffer , and bound material ( immunoprecipate ) removed by incubation with sds - page sample buffer . protein - g agarose beads were removed by centrifugation and the remaining supernatant resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ; 10%-20% tris - glycine , invitrogen , inc . ) . after electrophoresis , the resolved protein was transferred to a pvdf membrane and incubated overnight at room temperature in blocking buffer . the membrane was then incubated with a specific chicken rgs1 antisera purchased from prosci inc , ca ( 1  :  2000 dilution ) for two hours at room temperature , washed in blocking buffer , and incubated with a horseradish peroxidase ( hrp)-conjugated second antibody ( anti - chicken igy - hrp ; 1  :  1000 dilution , prosci inc , ca ) . immunoprecipated rgs1 was detected using a chemiluminescent detection method as previously described . after transfer to pvdf , transferred protein was also viewed using the reversible protein stain ponceau s ( thermo scientific , rockford , il ) . a human monocytic cell line ( thp-1 ) in active growth phase was incubated with or without ifn- ( 1000  iu / ml ) for 20 hours . cells ( 1  10/ml ) were collected , washed in cold pbs , and incubated on ice for 1 hour with a formyl peptide receptor ( fpr)-phycoerythrin or cxcr4-phycoerythrin - conjugated antibody ( fpr - pe or cxcr4-pe ) ( abcam , inc . apoptosis was determined by measuring the extent of expression of the apoptosis marker annexin - pe relative to the viable cell marker 7-amino - actinomycin d ( 7-aad ) . facs was performed using a beckman - coulter cytomic fc 500 mpl and cmp data analysis software ( beckman - coulter , fullerton , ca ) . calcein am - labeled ( molecular probes , carlsbad , ca ) primary monocytes and thp-1 cells were assessed for their ability to migrate using 5-m pore size multiscreen mic filter plates ( millipore , temecula , ca ) . one hundred fifty microliters of migration buffer ( hbss plus ca / mg , 0.1% bsa ) or buffer plus sdf1 , mcp1 , or mcp2 ( r&d systems , ca ) was placed in the bottom chamber . one hundred thousand ifn- or mock - treated cells in migration buffer ( 100  l ) were gently overlaid on the top chamber . the culture plates were incubated at 37c , 5% co2 for 24 hours , and cells migrating to the bottom chamber were quantified on a flexstation ii calibrated to measure endpoint fluorescence ( 488  nm excitation and 538  nm emission ) . results are shown as percentage of migration relative to the original number of cells applied . the mean values  standard deviations ( sd ) were determined for each given experiment . statistical tests comparing differences between groups were performed using the student 's t - test . a p - value of < .05 was considered to be significant . rgs1 is a member of a large family of regulators of g - protein signaling , the r4 rgs proteins . rgs1 is known to be expressed in immune cells , and overexpression in b - cells attenuates chemokine signaling . in this study , the specific induction of rgs1 by ifn- was first observed in pbmcs ( figure 1 ) . ifn- was shown to preferentially induce the expression of rgs1 over that of other rgs family members including rgs2 , rgs5 , rgs7 , rgs12 , and rgs14 ( figure 1(a ) ) . ifn--dependent induction of rgs1 was next determined in three purified immune cell populations isolated to homogeneity ( greater than 90% ) using negative selection . induction of rgs1 by ifn- was shown to occur in b cells , monocytes , and t cells ( figure 1(b ) ) . although all three cell types were responsive to ifn- stimulation , rgs1 induction appeared to be strongest in monocytes . pbmcs were stimulated for 4 or 18 hours with either ifn- , ifn- , or pma followed by determination of rgs1 rna expression ( figure 2 ) . results show a preferential induction of rgs1 by ifn- relative to both pma and ifn-. pma induction of rgs1 expression was also previously described in the human monocytic cell line u937 .   to confirm a direct role for ifn- in the induction of rgs1 , rgs1 expression was determined in the presence of neutralizing antibody that blocks activation of the ifn receptor ( figure 3 ) . the antibody concentration needed to neutralize the ifn receptor was determined using a cell line stably expressing an ifn - responsive reporter ( ht1080luc ) ( figure 3(a ) ) . ht1080luc cells incubated with neutralizing antibody showed a strong inhibition of rgs1 induction after ifn- stimulation relative to preimmune sera and untreated cells ( figure 3(b ) ) . these results demonstrate that the specific induction of rgs1 by ifn- is dependent upon activation of the ifn receptor . genetic risk factors for susceptibility to multiple sclerosis have recently been identified [ 30 , 31 ] , the most prominent being hla drb1 . interestingly , rgs1 was also included in one of the ms susceptibility loci identified . in this regard , it was important to determine if treatment of ms patients with ifn- leads to changes in rgs1 expression . based on previous pharmacokinetic studies , ms patients were asked to stop treatment for 64 hours to allow for a  washout  period in which ifn- dependent gene expression would return to baseline levels . patient blood was drawn just prior to administration of ifn- and at 4 , 18 , and 43 hours post - administration . pbmcs were isolated , rna purified and differential gene expression analyzed . as previously demonstrated , induction of a number of differentially expressed genes was observed in ms patients following administration of a single dose of ifn- . examination of the differential gene expression pattern using rma and sacred demonstrated a transient change in differential gene expression that returned to near preadministration levels by 42 hours . upon further examination , we observed the upregulation of rna encoding rgs1 in all seven ms patients studied ( figure 4 ) . this result supported our initial in vitro observations and demonstrated the regulation of rgs1 by ifn- in ms patients . in ms patients , the expression of rgs1 reached a maximum level 4 hours post - administration of ifn- and returned to near baseline by 42 hours . a similar time course of induction of rgs1 occurred in each patient ; however , patient - to - patient variability in the magnitude of response was observed ( figure 4 ) . variability of response was not due to differences in rna quality or method of genechip analysis . to validate the observed expression of rgs1 in ms patients , we performed an rgs1-specific taqman assay making use of the same rna samples used for the initial affymetric genechip analysis . using this approach , we also observed patient - to - patient variability in magnitude of response for the same samples , and a maximum expression of rgs1 at 4 hours post - administration of ifn- followed by a return to near baseline by 42 hours ( figure 5 ) . rna induction generally precedes that of protein expression although changes in rna half - life or stability could possibly alter the level of rna observed in the cell without resulting in an increased level of protein expression . therefore , demonstrating that the induction of rgs1 rna resulted in an increase in protein expression was important . the ability of rgs1 antisera to recognize rgs1 was initially determined by immunoblotting using purified 6xhisrgs1 ( figure 6 ) . 6xhisrgs1 was recognized by either rgs1 antisera ( figure 6(a ) , lane 2 ) or an antibody directed against 6xhistidine ( figure 6(a ) , lane 3 ) . pbmcs obtained from healthy individuals were stimulated with ifn- , ifn- , or pma for 20 hours . for normalization of protein levels , cell numbers were made identical and an equal protein concentration was used for each immunoprecipitation . cells were lysed and rgs1 was immunoprecipitated using an rgs1 polyclonal antisera . as shown in figure 6(b ) , rgs1 was strongly induced by ifn- stimulation and clearly present in immunoprecipitates from ifn-- and pma - stimulated cells ( figure 6(b ) , lanes 3 and 5 ) . rgs1 was not detected using preimmune sera ( figure 6(b ) , lane 1 ) and rgs1 was not appreciably detected in rgs1 immunoprecipitates using cell lysates made from pbmcs stimulated with ifn- ( figure 6(b ) , lane 4 ) . however , a faint protein doublet was observed in both unstimulated and ifn- stimulated cells ( figure 6(b ) , lanes 2 and 4 ) . this protein doublet likely represents the basal level of rgs1 expression in this cell population . the appearance of rgs1 as a protein doublet of approximately 23  kda is consistent with its expected molecular weight and previous observations . this may occur due to an amino acid truncation , misfolding of the protein , or the presence of protein isoforms . previous studies have documented the role of ifn- in immune modulation and expression of th1 and th2 cytokines [ 9 , 12 ] . less well documented is the role of ifn- in the regulation of immune cell migration , a process in which chemokines , gpcrs , and rgs proteins play a central role . while a limited number of studies have demonstrated that ifns induce shedding of adhesion molecules from the cell surface [ 32 , 33 ] , few studies describe a role for ifns directly in immune cell migration . in this regard , the upregulation of rgs1 by ifn- would be expected to result in a reduction in chemokine - dependent cell migration . to determine if such an association exists , we stimulated monocytes with ifn- for a time period demonstrated to sufficiently induce rgs1 ( figure 6 ) . a chemokine - dependent transmigration assay was then used to measure the degree of cell migration . both primary monocytes ( figure 7(a ) ) and the monocytic cell line thp-1 ( figures 7(b ) , 7(c ) , and 7(d ) ) were shown to migrate across an artificial barrier when stimulated with either sdf1 , mcp1 , or mcp2 . in all cases , stimulation of monocytes or thp-1 cells with ifn- for the time required to elevate levels of rgs1 inhibited monocyte transmigration relative to untreated cells . inhibition of chemokine - dependent cell migration by ifn- was dose dependent and consistent with the time and concentration required for inhibition of sdf1-dependent calcium flux ( data not shown ) . these results provide a direct measurement of the effect of ifn- on cell migration but only indirectly link the upregulation of rgs1 to regulation of cell migration . such effects could presumably give rise to indirect changes in gpcr levels and thereby influence rgs expression . therefore , it was important to show that ifn- used as described in these studies , did not influence gpcr expression or induce apoptosis . when using the apoptosis marker annexin , no difference in apoptosis was observed between untreated monocytes ( figure 8(a ) ) and those treated with ifn- ( figure 8(b ) ) . in terms of gpcr expression , cell - surface expression of two gpcrs ( cxcr4 and fpr ) representing different gpcr families analyzed . formyl peptide receptor ( fpr )   expression showed no difference in cell - surface expression in the absence ( figure 8(c ) ) or presence ( figure 8(d ) ) of ifn- treatment . a similar lack of affect of ifn- on the cell - surface expression of cxcr4   was also observed ( figures 8(e ) and 8(f ) ) . chemokines play a major role in the trafficking of immune cells to sites of inflammation , in and out of germinal centers located in lymphoid tissues , and in the repair of damaged tissue . migration of immune cells is mediated by a complex interplay between cell - surface adhesion molecules and chemoattractant receptors and ligands [ 3436 ] . cytokines , a family of immune modulators different from chemokines , play a central role in mediating both innate and adaptive immunity and protection from viral infection [ 12 , 34 , 35 ] . harmonizing the interplay between chemokines and cytokines appears essential for the establishment of an effective and properly regulated immune response . rgs proteins regulate chemokine - induced activation of gpcrs by accelerating the gtpase activity associated with the g - protein alpha subunit . the rgs protein family consists of 30 or more distinct proteins containing one or more rgs domains . in mammalian cells , rgs proteins interact with other molecules involved in signal transduction including receptors , phospholipids , and scaffolding proteins [ 1921 , 27 ] . some rgs members like rgs2 and rgs3 have a wide distribution of expression while others like rgs4 , rgs6 , rgs7 , rgs8 , rgs9 , rgs10 , rgs11 , and rgs14 are only found in a specific organ or tissue . rgs1 appears to be highly expressed in lymphocytes , suggesting a role for rgs1 in immune cell regulation . in support of this role , previous studies using rgs knockout mice suggest a strong link between rgs1 expression and b cell migration . results described in this study identify a possible additional role for ifn- in the regulation of immune cell function . our findings demonstrate that ifn- may influence chemokine - dependent signaling though induction of a negative regulator of g - protein signaling . regulating immune cell trafficking to sites of inflammation or , as in ms , inflammatory lesions in the central nervous system ( cns ) , goes hand in hand with immunological properties previously associated with type i ifns [ 9 , 12 , 32 , 3741 ] . previous studies have described a role for ifn- in regulating immune cell migration by enhancing shedding of adhesion molecules from the cell surface . ifn- regulation of rgs1 points to an additional means by which ifns can exert an inhibitory effect on chemokine - dependent lymphocyte migration . such a mechanism can help to explain the previously described involvement of ifns in the regulation of immune cell migration across human brain microvascular endothelial cell monolayers , the transverse of t cells through basement membranes , and human lymphocyte transmigration . it should be noted that ifn- regulation of rgs1 alone may not be sufficient to regulate the full extent of ligand - dependent gpcr activation of g - proteins . it is likely that other proteins interact with rgs1 or affect the regulation of additional rgs proteins to fine tune specific activation and migration of immune cells during inflammation . recent studies have demonstrated that upregulation of rgs1 reduces the migration of regulatory t - cells ( tregs ) relative to nave t cells . in this regard , g - protein regulation by rgs1 and other rgs family members can be highly cell - specific and involves other unidentified factors [ 46 , 47 ] . such specificity would also suggest that rgs control of immune cell responsiveness would be dependent on the presence of a given gpcr . translating in vitro observations made in cell culture to in vivo responses observed directly in a disease setting can provide valuable insights into the possible mechanism of action of a drug . in addition , the identification of an rgs1 genetic risk factor susceptibility locus in ms suggests a possible role for single - nucleotide - polymorphism - derived rgs1 variants in disease susceptibility . therefore , it is interesting to note that rgs1 is upregulated in ifn- treated ms patients perhaps in an effort to compensate for an abnormal rgs1 . formation of inflammatory lesions in ms patients as recognized by mri using gadolinium enhancement is a measurement of cns inflammation associated with ms . formation of lesions within the cns is thought to be mediated , in part , by the migration of activated immune cells from the periphery into the cns . once in the cns , they interact with local antigen - presenting cells including astrocytes and microglia to further drive formation of inflammatory lesions visible by mri . in addition , suppression of immune responses in ms patients has been linked to a reduction in the levels of oxidative stress , which , when left unchecked , can lead to neurodegeneration and axonal loss commonly observed in ms [ 49 , 50 ] . the observed in vitro upregulation of rgs1 by ifn- provided the opportunity to study the in vivo regulation of rgs1 directly in ms patients treated with ifn-. although only seven individual patents were studied , all patients showed significant upregulation of rgs1 expression after ifn- administration . the upregulation of rgs1 as measured by gene expression profiling in ms patients highlights the possible importance of rgs1 for the regulation of chemokine activity in the treatment of ms . a single study such as this is not sufficient to demonstrate rgs1 regulation by ifn- provides a new sole mechanism of action , nor is the effect likely unique to ms . however , such a mechanism could help to explain the observed inhibitory effects of ifn- on migration of activated immune cells from the periphery to sites of inflammation within the cns . ifn- inhibition of cell migration demonstrates a role for ifn- in the regulation of immune cell migration . rgs1 upregulated by ifn- would be expected to enhance immune cell migration by accelerating gtpase activity , negatively regulating g - protein coupled receptor signaling . ifn- was demonstrated to inhibit monocyte migration across an artificial barrier in response to chemokine . inhibition of cell migration coincided with the time required to upregulate rgs1 in immune cells , suggesting an association between inhibition of cell migration and expression . unfortunately , we were unsuccessful in identifying an sirna able to downregulate rgs1 expression in these cells . therefore , a direct link between ifn- induced rgs1 and cell migration will require further studies . a rgs1 murine knockout is available and could provide an additional means to studying the role of ifn- in regulation of rgs1 and cell migration . ifn- did not appear to affect gpcr receptor expression or induce apoptosis in immune cells using conditions described in this study . therefore , the observed reduction in monocyte cell migration was not due to an induction of cell death or a loss of g - protein coupled receptors from the cell surface . the observation that a cytokine such as ifn- can influence chemokine - dependent g - protein activation suggests a possible mechanism for crosstalk between these two important families of immune regulators . studies are underway to further understand the relationship between ifn- , gpcrs and rgs proteins with regard to control of immune cell surveillance , maturation , migration , and inflammation .
we present evidence of a link between interferon-1b ( ifn- ) and g - protein signaling by demonstrating that ifn- can induce the expression of the negative regulator of g - protein signaling 1 ( rgs1 ) . 
 rgs1 reduces g - protein activation and immune cell migration by interacting with heterotrimeric g - proteins and enhancing their intrinsic gtpase activity . in this study , 
 ifn- treatment resulted in the induction of rgs1 in peripheral blood mononuclear cells ( pbmcs ) , monocytes , t cells ,   and b cells . 
 induction of rgs1 by ifn- was concentration dependent and observed at both the rna and protein level . 
 other members of the rgs family were not induced by ifn- , and induction of rgs1 required the activation of the ifn receptor . 
 in addition , rgs1 induction was observed in pbmcs obtained from ifn--treated multiple sclerosis patients suggesting a possible , as yet unexplored , involvement of g - protein regulation in disease treatment .   the upregulation of rgs1 by ifn- has not been previously reported .