document: glucocorticoids have been extensively characterized as being anti - inflammatory   and/or immunosuppressive   in therapeutic settings and are generally discussed as opposed to the development of inflammation and limiting of production or maturation of some effector immune cell types , in a physiological context . 
   nevertheless , there is a well - established association between stress and allergic diseases , including asthma [ 4 , 5 ] . 
   glucocorticoids , which are an essential part of systemic stress responses , play coadjuvant roles in promoting inflammation and may promote th2-type immunity through differential effects on th1  th2 cytokine production [ 5 , 6 ] . chronically stimulated eosinophil production ( eosinopoiesis ) is an important feature of human asthma   and of murine allergic asthma models . in both cases , allergen challenge of sensitized subjects increases eosinopoiesis in the bone - marrow [ 7 , 8 ] . 
   this effect is antigen - specific and can be abolished by inducing oral tolerance to the allergen , which affects both eosinophils and neutrophils in bone - marrow . 
   the effects of oral tolerization in bone - marrow neutrophil and eosinophil granulocytes can be duplicated by transfer of splenic t lymphocyte subpopulations from tolerized / sensitized / challenged donors to histocompatible naive recipients . 
   these observations highlight the importance of acquired cellular immunity in regulating the hematological response to allergen sensitization and challenge . 
   they further suggest the possibility that granulopoiesis , encompassing both eosinophil and neutrophil production , might be regulated by lymphocyte populations in nonsensitized subjects as well . in an allergic asthma model , we demonstrated a critical role for endogenous glucocorticoids in the hematological response to allergen challenge : challenge induces a corticosterone surge that is paralleled by increased eosinophilia of bone - marrow in vivo and by increased responsiveness to il-5 , the major eosinopoietic cytokine , ex vivo ; the bone - marrow response to challenge is abolished by blockade of glucocorticoid signaling or glucocorticoid production . on the other hand , in the absence of allergen sensitization and challenge , we have also obtained evidence of a link between the corticosterone surge induced by mild surgical trauma and short - term bone - marrow eosinophilia ; again , blockade of endogenous glucocorticoid production or action abolished the bone - marrow eosinophilia induced by trauma . 
   these in vivo effects of corticosterone , an endogenous glucocorticoid released by the adrenal glands , are paralleled by those of exogenously provided corticosterone or dexamethasone on murine bone - marrow [ 12 , 13 ] and of other glucocorticoids on human hemopoietic cells . in balb / c mice , 
   dexamethasone increases eosinophil production in murine bone - marrow culture [ 12 , 13 ] and primes bone - marrow cells in vivo for increased ex vivo responses to il-5 . during further screening of inbred mouse strains for differences in the granulopoietic responses to dexamethasone , we observed bone - marrow eosinophilia in mice of the c57bl/6 ( b6 ) background injected with dexamethasone , which was undetectable in perforin - deficient b6 mutants   submitted to the same treatment . 
   perforin is a major mediator of cellular immunity [ 1519 ] , expressed in lymphocyte populations which fight viral   and bacterial pathogens [ 16 , 17 ] as well as malignant cells   in the context of both innate and acquired immune responses . 
   perforin is also expressed by murine bone - marrow neutrophils , which have a critical regulatory role in t cell - mediated contact hypersensitivity . 
   perforin deficiency is known to induce complex changes in leukocyte populations in humans and mice [ 16 , 17 , 20 ] , including a classical presentation of familial hemophagocytic lymphohistiocytosis , characterized by early life onset , high mortality , and multiple immunological defects , including uncontrolled activation and proliferation of cd4 + and cd8 + t cells , cytokine storm , macrophage activation and proliferation , pancytopenia , and anemia . here 
   we report that perforin deficiency also presents a selective defect in granulocyte production , which can be corrected by wild - type lymphocyte transfer . 
   sh30088.03 ) , rpmi1640 ( sh30011.01 ) , and imdm ( sh30228.01 ) were from hyclone ( logan , ut , eua ) ; l - glutamine ( g7513 ) , penicillin - streptomycin solution ( p4333 ) , essential amino acids solution ( 50x ) ( m5550 ) , methylcellulose ( m0387 ) , dexamethasone ( 21-phosphate , disodium salt , d1159 ) , mifepristone ( ru486 , m8046 ) , and histopaque-1083 ( 10831 ) were from sigma - aldrich corporation ( st . louis , mo , eua ) ; agar noble ( 0142 - 15/21422 ) was from difco ( detroit , mi , eua ) ; nonessential amino acids solution ( 100x ) , ( 11140 - 050 ) and mem vitamin solution ( 100x ) ( 11120 - 052 ) were from gibco life technologies ( carlsbad , ca , usa ) ; recombinant murine il-5 ( 405-ml-025 ) was from r&d systems ( minneapolis , mn , usa ) ; recombinant murine gm - csf ( 315 - 03 ) was from preprotech ( rocky hill , nj , usa ) ; rat antimouse cd8b ( clone : ebioh35 - 17.2 , 12 - 0083 - 82 , 0.2  mg / ml ) was from ebioscience ( san diego , ca , usa ) ; magnetic microspheres conjugated to antimouse cd4 ( l3t4 , 130 - 049 - 20 ) and to goat antirat igg ( 130 - 048 - 501 , as secondary antibody to primary rat antimouse cd8b ) were from miltenyi biotec ( ambriex , sp , brazil ) . 
   wild - type c57bl/6 , perforin - deficient ( pfp ) mutants of the b6 background , and wild - type b6.129 mice were bred by cecal - fiocruz , rio de janeiro , brazil . 
   the pfp stock was derived from the original b6.129s6-pfp stock   by backcrossing to c57bl/6 at fiocruz . 
   unless otherwise indicated , the wild - type controls for the experiments shown were c57bl/6 ; in selected experiments , b6.129 wild - type controls were used and yielded the same results as c57bl/6 ( not shown ) . 
   the animals were housed and handled following institutionally approved guidelines under license l-00209 from ceua - fiocruz . 
   routinely , female mice were used for the experiments , since male mice characteristically fight for dominance in the same cage , and the stress associated with fighting may confound the interpretation of the results . 
   we have no evidence , however , that the granulopoietic responses described here , including the strain differences , are restricted to females . dexamethasone in saline solution was injected i.p . 
   ( 200  l , i.p . , amounting to 5  mg / kg ; ) . 
   ru486 in 0.1% methylcellulose was given intragastrically 2  h before dexamethasone with a gavage needle ( 200  l , amounting to 100  mg / kg ) . 
   for lymphocyte transfers , 10 nylon - wool purified cells   from naive c57bl/6 donors in a 100  l volume of sterile saline were injected into the tail vein of pfp recipients , once . 
   controls received an equal number of cells from pfp donors . where lymphocytes depleted of cd4 + or cd8 + cells were used , the amount of cells injected was that recovered from 10 initial unseparated lymphocytes , in 100  l sterile saline , i.v . 
   dexamethasone was administered to the recipients , in both cases , 48  h after lymphocyte transfer . 
   where indicated , bone - marrow , peripheral blood from the abdominal vena cava , spleens , and whole thymuses were collected , for enumeration of cells or determination of relative weight of the thymus ( mg / g body weight ) [ 21 , 23 ] . 
   bone - marrow collected from both femurs of each mouse in 5  ml rpmi1640 medium/1% fbs with a 22-gauge needle and kept on ice was used for total and differential counts and cell culture ( see below ) . 
   spleen mononuclear cells were the source for isolation of lymphocytes ( see below ) [ 24 , 25 ] . 
   differential counts were carried out in cytocentrifugates after fixation in pbs-10% formaldehyde and staining for eosinophil peroxidase ( epo [ 26 , 27 ] ) followed by counterstaining with harris ' hematoxylin . 
   liquid bone - marrow cultures were established at 37c , with 10 bone - marrow cells in 1  ml rpmi1640/10% fbs , in 5% co2/95% air , plated in 24-well plates ( cat . 
   n : 142475 , nunc brand products ) with il-5 ( 1  ng / ml ; 7 days ) . where indicated , dexamethasone was added ( 10  l / well , to 10  m final concentration ) . 
   absolute numbers were calculated from total cell counts in turk 's solution multiplied by the % of epo+ cells ( eosinophil - lineage cells , both mature and immature ) in cytocentrifuge smears . 
   triplicate semisolid ( clonal ) bone - marrow cultures were established for 7 days with 2  10 cells adjusted to 1  ml of 1  :  1 imdm / agar mix , in 35  mm tissue culture plates ( nunc ) , in the presence of gm - csf ( 2  ng / ml final ) , with or without dexamethasone ( 10  m ) , at final 20% fbs and 0.3% agar concentrations . 
   total colonies ( defined as aggregates > 50 cells derived from a single progenitor cell ) [ 8 , 12 ] were scored at day 7 under an inverted microscope ( 400x , phase contrast ) . for lymphocyte isolation , spleens were collected and minced in rpmi1640/1% fbs on tissue culture plates . 
   spleen mononuclear cells ( 2  10 in 10  ml rpmi1640/1% sfb ) were isolated by centrifugation on a 1.083-density ficoll - hypaque cushion ( 3  ml , at 400  g , for 30 minutes , at room temperature , following manufacturer 's instructions ) [ 24 , 25 ] . 
   cells recovered from the medium / ficoll - hypaque interface were collected , washed in serum - free medium , resuspended , counted , and further separated on nylon - wool columns [ 24 , 25 ] at 4  10 cells/2  ml / g nylon wool . 
   cells eluted in a total 25  ml warm medium , dropwise , were washed and counted before cytocentrifugation / staining or incubation with antibodies . 
   depletion of cd4 + cells was done with 10  l l3t4/10 nylon - wool purified lymphocytes in 100  l serum - free rpmi1640 , on ice , following manufacturers ' instructions . 
   depletion of cd8 + cells was in two steps , with rat antimouse cd8b conjugated with pe , followed by goat antirat igg ( 20  l/10 lymphocytes in 80  l medium , on ice ) . 
   columns were eluted with 9  ml medium , dropwise , over a 5-minute period . 
   the numbers of experiments ( n ) are indicated in the caption of the figures , to avoid overcharging the figures and captions . for comparisons of two groups ( figures 1 and 2 ) , we used the two - tailed t - test with separate variances ( systat for windows , version 5 , systat inc . , 
   evanston , il ) . for multiple comparisons ( figures 3 , 4 , 5 , and 6 ) 
   , we used anova , with the tukey hsd correction for groups of equal size ( systat for windows ) or with bonferroni 's correction for groups of unequal size ( using prisma 5 for windows , graph pad , la jolla , ca ) , unless otherwise indicated in section 3 . 
   following preliminary experiments ( not shown ) that evidenced a positive response to dexamethasone in b6 wild - type mice , as well as the absence of any response , positive or negative , in pfp mutants of the b6 background , we reviewed data on freshly harvested bone - marrow from a large number of mice of both strains ( n = 44 and n = 48 , resp . ) , to look for evidence of strain differences in bone - marrow steady - state parameters , in the absence of dexamethasone exposure ( figure 1 ) . 
   a significant difference was observed in this large series , with pfp mice having lower bone - marrow cellularity than wild - type controls ( figure 1(a ) ; p < 0.001 ) 
   . the data available for epo+ cells ( figure 1(b ) ;  p = 0.033 ) and neutrophils ( figure 1(c ) ; p = 0.010 ) of the mice in the large series showed significant differences as well , with lower counts in pfp mutants . 
   as the two groups were defined only on the basis of genetic differences ( presence or absence of functional perforin genes ) , these differences could still be accounted for , in principle , by variance due to nongenetic factors within these groups , especially those which influence growth and development . because bone - marrow cellularity is roughly proportional to the size of the animal 
   , we next evaluated whether these differences would disappear in the comparison between groups of control and mutant mice matched by weight ( figures 1(d)1(f ) ) . 
   significant differences were still observed for total cells ( figure 1(d ) ; p = 0.001 ) , eosinophils ( figure 1(e ) ; p = 0.004 ) , and neutrophils ( figure 1(f ) ; p = 0.011 ) , all three parameters being lower in the mutant mice . because age might affect bone - marrow function through mechanisms unrelated to body weight gain ( as senescence may have an earlier onset in some strains ; furthermore , age is a major determinant of incidence of many pathological processes , such as malignancies ) , we next examined whether matching by age ( all animals at 12 weeks ) would eliminate the differences . 
   significant differences were still found for all three parameters ( for figures 1(g)1(i ) , resp . 
   , p = 0.001 , p = 0.006 , p = 0.001 ) , which were lower in the mutant mice . even matching by both weight and age ( for figures 1(j)1(l ) , resp . 
   , p = 0.022 , p = 0.032 , p = 0.001 ) failed to eliminate these significant differences between controls and mutants for any of the three parameters , which were all lower in the mutant mice . 
   overall , this suggests that bone - marrow cellularity , as well as bone - marrow eosinophil and neutrophil counts , is significantly lower in pfp mutants than in b6 mice and that this difference can not be dismissed as created by undue comparisons of two groups differing in body weight , age , or both . 
   we further examined ( figure 2 ) the counts of total cells , lymphocytes , neutrophils , and eosinophils in peripheral blood of weight - matched ( median 21  g , range 1923  g ) b6 and pfp mice . unlike bone - marrow 
   , peripheral blood total nucleated cell counts did not differ significantly between these strains ( figure 2(a ) , p = 0.795 ) , nor did lymphocyte counts ( figure 2(b ) , p = 0.417 ) . 
   by contrast , both neutrophil ( figure 2(c ) , p = 0.025 ) and eosinophil ( figure 2(d ) ; p = 0.030 ) counts were significantly different , and , like in bone - marrow , lower in pfp mice . 
   together , the data in figures 1 and 2 suggest that , even in the absence of dexamethasone , pfp mice have reduced granulocyte numbers both inside and outside of bone - marrow , relative to wild - type controls of comparable body weight . 
   our original observation of this strain difference was increased % eosinophils in cultured bone - marrow from b6 , but not pfp , mice when both il-5 and dexamethasone were present ( jones and cardoso de mendona , unpublished observations ) . 
   this is , however , insufficient to characterize a strain difference in response to dexamethasone , because the % of a given cell type in a bone - marrow sample , which has a highly heterogeneous composition , can be increased artifactually by a corresponding decrease in another cell type , rather than by a positive effect of dexamethasone on the cell type of interest . to confirm a positive effect of il-5 and dexamethasone in stimulation of wild - type and mutant eosinopoiesis , 
   liquid cultures were established from the same number of bone - marrow cells ( 10 ) from b6 or pfp donors , in the presence of il-5 , alone or associated with dexamethasone , and the absolute numbers of eosinophils in the culture were determined . 
   as shown in figure 3 , eosinophils were produced in cultures of il-5-stimulated wild - type and mutant bone - marrow ( figure 3(a ) ) . 
   control cultures lacking il-5 do not contain eosinophils at the end of the culture period ( not shown ; ) . 
   counts of epo+ cells recovered at the end of the culture were increased in the presence of dexamethasone ( 10  m ) , relative to il-5 controls , in cultures from b6 controls , but not from pfp mutants ( figure 3(a ) , p < 0.001 ) . nevertheless , the il-5 present was sufficient to sustain eosinopoiesis in the absence of dexamethasone by pfp bone - marrow to the same level observed in b6 control cultures . 
   on the other hand , data from semisolid cultures , which allowed us to examine the effects of dexamethasone on several classes of progenitors ( colony - forming cells ) , including the granulocyte ( g ) and granulocyte - macrophage ( gm ) progenitors , which produce neutrophils , are also shown in figure 3 . 
   the total counts of colonies formed by gm - csf - stimulated pfp bone - marrow progenitors , in the absence of dexamethasone , also differed significantly from those of b6 controls ( p  0.001 ) : even though identical numbers of bone - marrow cells were plated , less colonies were made by perforin - deficient bone - marrow ( figure 3(b ) ) . 
   furthermore , dexamethasone ( 10  m ) significantly stimulated colony formation by b6 bone - marrow ( p = 0.002 ) but failed to stimulate ( p = 0.558 ) pfp bone - marrow in the same conditions ( figure 3(b ) ) . 
   these observations show that bone - marrow progenitors from pfp mice differ significantly from those of b6 controls , because they form less colonies and do not respond to dexamethasone , which significantly enhances colony formation in the wild - type cultures . 
   we next examined the effect of dexamethasone ( 5  mg / kg injection ) on bone - marrow eosinophils and neutrophils , as well as on the relative weight of the thymus , which is significantly reduced by glucocorticoids . 
   as shown in figure 4 , dexamethasone injection increased the numbers of bone - marrow eosinophils ( figure 4(a ) , p = 0.003 ) and neutrophils ( figure 4(b ) , p = 0.019 ) in vivo , relative to saline - injected controls , in wild - type b6 mice , but not in pfp mice . 
   the effect of dexamethasone on eosinophil numbers in wild - type b6 mice was abolished by ru486 pretreatment , while ru486 had no effect of its own in the absence of dexamethasone , in either b6 or pfp mice ( not shown ) . 
   the relative weight of the thymus was significantly different ( p = 0.010 ) between body weight - matched pfp mice and wild - type controls , suggesting a decreased thymic cellularity in mutant thymus , even without exogenous glucocorticoid administration ( figure 4(c ) ) . 
   importantly , dexamethasone injection did reduce significantly the relative weight of the thymus in both wild - type ( p = 0.007 ) and mutant ( p = 0.001 ) mice ( figure 4(c ) ) . 
   again , the effect of dexamethasone on the thymus was abolished in both cases by ru486 pretreatment ( not shown ) . 
   together , these observations show that pfp bone - marrow is unresponsive to dexamethasone stimulation in vivo , although dexamethasone significantly increases eosinophil and neutrophil numbers in the bone - marrow of wild - type controls . 
   they also show that lack of responsiveness to dexamethasone in pfp bone - marrow is not due to a general lack of response to glucocorticoids , because the thymus of pfp mice responds to dexamethasone injection as expected . 
   because perforin is mainly expressed in various effector / regulatory lymphocyte subsets , it was important to test whether the perforin - related defect in bone - marrow response to dexamethasone could be corrected by introducing b6 lymphocytes in pfp mice . 
   we have done so with splenic lymphocytes from naive b6 donors , because both the baseline granulopoiesis defect and the defective granulopoietic response to dexamethasone were observed in the absence of allergic sensitization . the number ( 10 ) of lymphocytes for transfer into individual mice 
   was defined on the basis of similar reconstitution studies . to define whether elimination of a particular lymphocyte subpopulation defined by standard surface markers ( cd4/cd8 ) prevented reconstitution of the dexamethasone response , 
   10 total lymphocytes purified from spleen were submitted to alternative depletion protocols with marker - specific microbeads , and the depleted cells in the column effluent , corresponding to the number of cells negative for the selection marker present in the original lymphocyte sample , were injected . as shown in figure 5 , pfp recipients of wild - type lymphocytes ( unseparated ) show a significant in vivo response to dexamethasone injection , by increased total cell ( figure 5(a ) ; p = 0.032 , t - test ) , eosinophil ( figure 5(b ) ; p = 0.04 ) , and neutrophil ( figure 5(c ) ; p < 0.001 ) counts in the bone - marrow , sharply contrasting with observations in pfp mice in the absence of wild - type cell transfer ( compare figure 4 ) . on 
   the other hand , depletion of cd4 + or cd8 + cells in the wild - type lymphocyte preparation gave distinct results in the reconstitution assay , depending on the granulocyte population examined : while depletion of either subset abolished the ability of splenic lymphocytes to reconstitute the eosinopoietic response to dexamethasone ( figure 5(b ) ) , depletion of cd4 + cells did not prevent reconstitution of the neutropoietic response ( figure 5(c ) ; p = 0.013 ) , while depletion of cd8 + cells prevented reconstitution . as a further control , we performed transfer of pfp lymphocytes into pfp recipients and obtained no reconstitution of dexamethasone responses , judged by any of these three parameters ( figure 6 ) . in these controls , 
   dexamethasone reduced significantly the relative weight of the thymus , showing that dexamethasone was able to reach systemically active levels even if bone - marrow showed no evidence of responding to it . 
   overall , these observations suggest that lymphocytes from the spleen of naive wild - type mice , but not pfp mice , are able to reconstitute in the short term the granulopoietic responses to dexamethasone , but different cells may be involved in reconstitution of lineage - specific ( eosinophilic versus neutrophilic ) responses . 
   this is , to our knowledge [ 1520 ] , the first description of a selective defect in granulopoiesis associated with perforin deficiency in mice and of its correction by the intravenous transfer of wild - type lymphocytes . as such 
   , it extends the range of manifestations associated with perforin deficiency and raises the issue of how perforin contributes to regulation of granulocyte lineages in vivo . because the wild - type lymphocyte populations interact with a drug ( dexamethasone ) which initiates signaling through the ru486-inhibitable glucocorticoid receptor , to stimulate in vivo eosinophil and neutrophil production 
   , these observations may be relevant to processes in which bone - marrow is stimulated by immune responses   or trauma   with involvement of the same receptor and of endogenous glucocorticoids released by the adrenal glands . 
   perforin is a well - characterized effector protein , mainly ( but not exclusively ) expressed in lymphocytes which share the ability to induce cell - mediated cytotoxicity [ 1520 ] . 
   perforin deficiency was initially characterized by the loss of important cytotoxic lymphocyte functions [ 1520 ] . 
   however , it was soon realized that perforin deficiency entails other , more complex , functional consequences , leading to the development of type ii familial hemophagocytic lymphohistiocytosis [ 16 , 17 , 20 ] , which shares pathophysiological features with the macrophage activation syndrome , including interferon- ( ifn- )  overproduction . 
   hence , perforin deficiency has broader consequences in addition to impairment of cytotoxic lymphocyte function , and ifn- , alone or in association with other  cytokine storm  components , may transduce its effects on other leukocyte lineages . 
   one of the issues raised by our findings is whether the lower granulocyte numbers in blood and bone - marrow of pfp mice are due to the progression of the murine equivalent of the human familial hemophagocytic lymphohistiocytosis [ 16 , 17 , 20 , 28 ] . 
   it should be noted that the human familial hemophagocytic lymphohistiocytosis is a severe condition , presenting early in life in most cases and associated with important mortality [ 20 , 28 ] . 
   also , phenotypically distinct forms correspond to different mutations with perforin expressed at lower levels and/or with altered properties , a situation that is not applicable to the present study , which employed perforin null mice . the perforin - deficient mice in the original walsh et al . 
   study ( from which the mice used in this study descend ) were described as healthy for at least 5 months . in our study , mice were certified spf and maintained in microisolator units , with no evidence of mortality , stunting , or signs of infection during the entire observation period of up to 3 months ( 12 weeks ) . on the other hand , pfp defect in response to dexamethasone 
   could be corrected by transfer of as few as 10 lymphocytes 48  h before the stimulus . 
   the prompt reconstitution of this defect argues against the lack of dexamethasone response in pfp animals being caused by a chronic inflammatory disease of the bone - marrow , such as familial hemophagocytic lymphohistiocytosis , which in humans is challenging for therapy . 
   because correction was achieved by simple transfer of lymphocytes from naive donors , we think it is more likely that the transferred lymphocytes provided something that was lacking in the recipient . 
   neutrophils were reduced in bone - marrow and blood of pfp mice relative to b6 controls . 
   this not only shows that the neutrophil deficiency has peripheral expression but further argues against reduced numbers in bone - marrow being due to an increased export of neutrophils to the periphery . 
   the evidence from colony - forming assays suggests , instead , that production is greatly reduced in pfp relative to b6 mice , as there were less gm - csf - responsive progenitors in an identical number of bone - marrow cells . 
   reduced production would explain the decrease in granulocyte counts both outside and inside of bone - marrow . 
   on the other hand , normal numbers of lymphocytes and total leukocyte counts show that the defect is selective for granulocytes , which are not the major circulating leukocyte subpopulation in mice , so that reduction in their numbers does not have a major impact on total circulating leukocyte counts . 
   neutropenia is associated with increased susceptibility to bacterial and fungal infection , provided it is severe enough . 
   this was not observed in our study , so presumably the pfp mice were capable of coping with the microorganisms present in a rather clean environment ( spf conditions , microisolator housing ) . 
   it remains to be seen , however , whether following a more severe infectious exposure , such as that associated with sepsis induction , pfp mice would prove more vulnerable to bacterial dissemination . 
   this will be addressed in future studies , since emergency granulopoiesis , as opposed to baseline granulopoiesis , is driven by gm - csf and related hemopoietic cytokines . 
   the lack of appropriate response to gm - csf , both in the absence and in the presence of dexamethasone , would predict that emergency granulopoiesis would be defective in pfp mice and might therefore negatively influence the outcome of sepsis . 
   the hypothalamus - pituitary - adrenal axis is activated in sepsis , and therefore it is possible that emergency granulopoiesis is driven by endogenous glucocorticoids , in a way consistent with our observations . 
   the prediction , in this case , is that perforin would be required for normal host response to bacterial sepsis through an effect on glucocorticoid - mediated signaling in the bone - marrow . 
   the ability of lymphocyte preparations to reconstitute responses to dexamethasone was abolished by depletion of cd4 + and/or cd8 + cells before transfer , depending on the granulocyte population . for neutrophils , cd8 
   while this suggests that different mechanisms are involved for eosinophils and neutrophils , it certainly raises the issue of the lymphocyte subpopulation involved . since naive lymphocytes are sufficient , it is not likely that conventional t cells , cd4 + or cd8 + , are responsible , for they would not be activated in this isolation protocol [ 24 , 25 , 31 ] . 
   conventional nk cells would not be a strong candidate , since cd4 + or cd8 + lymphocyte depletion would probably not eliminate them [ 32 , 33 ] . 
   innate lymphocytes other than conventional nk cells , however , would be expected to be present in naive mouse spleens [ 32 , 33 ] . 
   natural killer t cells , which include subsets expressing cd4 and cd8 , are found in significant numbers in b6 spleen , present an activated memory phenotype without known activating exposure , and exert multiple regulatory functions through rapid secretion of large amounts of cytokines [ 32 , 33 ] , might perhaps account for some of our observations . 
   regardless of innate or adaptive lymphocyte population involved , two questions which require further study are as follows : ( a ) is perforin the missing component that is brought by the transferred lymphocytes ? and ( b ) do these lymphocytes act by homing to the recipients ' bone - marrow and interacting locally with granulocyte progenitors / precursors ? both questions , however , require a more complex experimental design and resources to allow us to detect and modify perforin expression inside living lymphocytes , as well as monitor their homing to the bone - marrow compartment . for this reason , 
   such experiments fall outside the scope of this paper , which is limited to the demonstration of the defect and of its correction . finally , it should be noted that lymphocyte transfer restored the short - term response to dexamethasone but did not bring the baseline counts of eosinophils and neutrophils to the same level of the b6 controls . 
   this might be interpreted as evidence that steady - state granulopoiesis and response to dexamethasone are unrelated processes ( i.e. , their perforin deficiency entails two distinct defects in granulopoiesis , rather than one defect with distinct manifestations ) . 
   we do not support this interpretation , however , because the duration of the experiment might be insufficient for an effect of lymphocyte transfer alone on steady - state granulocyte numbers to become noticeable . 
   it is also possible that demonstration of a durable effect on steady - state granulocyte numbers would require not only an extended observation period but also an increased or repeated input of wild - type lymphocytes and an interaction of exogenous lymphocytes with endogenous adrenal glucocorticoids as well .
summary: exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone - marrow . 
   a hematological response dependent on endogenous glucocorticoids underlies bone - marrow eosinophilia induced by trauma or allergic sensitization / challenge . 
   we detected a defect in granulopoiesis in nonsensitized , perforin - deficient mice . 
   in steady - state conditions , perforin- ( pfp- ) deficient mice showed significantly decreased bone - marrow and blood eosinophil and neutrophil counts , and colony formation in response to gm - csf , relative to wild - type controls of comparable age and/or weight . 
   by contrast , peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency . 
   dexamethasone enhanced colony formation by gm - csf - stimulated progenitors from wild - type controls , but not pfp mice . 
   dexamethasone injection increased bone - marrow eosinophil and neutrophil counts in wild - type controls , but not pfp mice . 
   because perforin is expressed in effector lymphocytes , we examined whether this defect would be corrected by transferring wild - type lymphocytes into perforin - deficient recipients . 
   short - term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild - type donors . 
   transfer of the same amount of splenic lymphocytes from perforin - deficient donors was ineffective . 
   this demonstrates that the perforin - dependent , granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations .