eosinophils are a minority granulocyte population , which contributes to the pathophysiology of allergic inflammation , hypereosinophilic syndromes , and some malignancies [ 14 ] . a role for eosinophils in resistance to multicellular ( helminth ) parasites has long been proposed , based on the strong association of blood and tissue eosinophilia with worm infections and on the evidence that eosinophils can damage or kill helminths , in specific experimental conditions [ 5 , 6 ] . nevertheless , a generally protective in vivo role for eosinophils against worm infections remains elusive , partly because host responses to multicellular parasites represent a compromise between the competing needs to reduce parasite burden and to limit immune - mediated tissue damage , to which eosinophils significantly contribute [ 7 , 8 ] . alternatively , mechanisms through which eosinophils may directly fight infection by various classes of microbial ( bacterial , fungal , protozoal , or viral ) pathogens include secretion of antimicrobial defensin - like proteins ; release of sticky cellular contents that capture pathogens , closely resembling neutrophil extracellular traps ; secretion of halogen microbicidal derivatives ; release of enzymes with antiviral activity and other roles in innate immunity [ 12 , 13 ] ; and secretion of a wide array of immunoregulatory cytokines . while the contribution of eosinophils to immunity as directly antimicrobial effector cells is likely limited by their scarcity , they could be helpful in conditions in which neutrophil access or macrophage function would be reduced : for neutrophils , tissue entry is restricted in normal conditions ; regarding macrophages , their microbicidal effector function is highly dependent on appropriate activating signals , including cytokines . by contrast , eosinophils are far more numerous in normal tissues than in blood and home to mucosal interfaces with the environment [ 24 ] , which represent potential gateways for microbial infection . they are a source of numerous immunoregulatory cytokines   and lipid mediators , which might play a role in recruitment / activation of other leukocyte subtypes . because of the scarcity of eosinophils , many important observations were made in conditions in which their numbers are already increased , due to allergic sensitization or experimental helminth infection , such as the discovery of eotaxin ( ccl11 ) , a chemoattractant that induces eosinophil accumulation in the skin of sensitized ( i.e. , eosinophilic ) guinea pigs [ 2 , 3 , 18 ] . while other potent eosinophil chemoattractants , such as pgd2 [ 19 , 20 ] and oxo - ete , have also been characterized , many factors reinforce the current understanding of eotaxin as a specialized chemoattractant which acts primarily on granulocyte subtypes relevant to allergy and worm infections [ 24 ] . these factors include the reported selectivity of eotaxin for the eosinophil [ 4 , 17 , 22 , 23 ] and basophil [ 20 , 24 , 25 ] lineages , and its interaction with hematopoietic cytokines , such as il-5 [ 26 , 27 ] and gm - csf , which promotes eosinophil production in bone marrow   and extramedullary sites , ultimately inducing blood and tissue eosinophilia . the alternative view , namely , that eotaxin is part of a broader regulatory network comprising multiple cell populations in addition to eosinophils and basophils , is also suggested by observations of a wide variety of eotaxin effects , including its ability to attract neutrophils and macrophages   and smooth muscle cells . eotaxin , also produced by fibroblasts [ 30 , 31 ] , has been associated with fibrotic processes in several settings [ 32 , 33 ] . within this wider framework , we have reexamined whether , in a nonsensitized host , eotaxin would recruit other leukocyte populations besides eosinophils and basophils and further examined whether its effects were dependent on 5-lipoxygenase ( 5-lo ) , the key enzyme in the leukotriene production by eosinophils [ 17 , 27 ] . the evaluation of both aspects was prompted by observations in mice which develop eosinophilia in response to subcutaneously implanted insoluble antigen pellets . while i.p . challenge of implant recipients with soluble allergen selectively recruited eosinophils to the peritoneal cavity , this effect was blocked by the 5-lo - activating protein inhibitor , mk886 , and duplicated by the 5-lo product , ltb4 , neither of which is eosinophil - selective . importantly , eotaxin , which duplicated the effects of allergen , was equally blocked by mk886 . equally unexpected was the failure of ltb4 , a potent neutrophil chemoattractant , to recruit neutrophils , while it effectively attracted eosinophils in this allergic model . these observations raised the possibility that the eosinophil selective effect of both chemoattractants ( eotaxin and ltb4 ) observed in vivo was dependent on the host being sensitized . we tested this hypothesis for eotaxin first , by examining its effects in a nave host , as well as the effect of 5-lo blockade on the effectiveness of eotaxin . we report that eotaxin recruits a mixed leukocyte population to the peritoneal cavity of nave mice and provide evidence of essential roles for both 5-lo and eosinophils in the accumulation and functional activation of neutrophils in this model . rpmi 1640 medium ( sh30011.01 ) and fetal calf serum ( sh30088.03 ) were from hyclone ( logan , ut ) ; penicillin 100  u / ml ( pen - b ) , streptomycin 100  mg / ml ( s9137 ) , ovalbumin ( grade ii and grade iv ) , isotonic percoll , and histopaque density 1.083 solution from sigma - aldrich ( st . louis , mo ) ; recombinant murine eotaxin ( 250 - 01 ) from peprotech ( rocky hill , nj ) ; and recombinant murine il-5 from r&d ; mk-886 ( 475889 ) 1  mg / kg from cayman chemicals ( ann arbor , mi ) , dissolved in 0.1% methylcellulose , was given as an intragastric bolus in a 0.2  ml volume . rat anti - murine eotaxin monoclonal neutralizing antibody ( clone 42285 ) and rat anti - murine igg2a control monoclonal antibody of matched isotype ( clone 54447 ) were from r&d ( minneapolis , mn ) . inbred mice , male and female , aged 810 weeks , provided in spf condition by cecal - fiocruz ( rio de janeiro ) , were of the following strains : balb / c ; alox ( 5-lo - deficient ) and pas-129 ( wild - type control of the same background ) ; and balb / c mutants lacking an enhancer element in the promoter region of gene coding for the gata-1 transcription factor , required for eosinophil lineage determination ( gata-1 mice , for short ) . animal housing , care , and handling followed institutionally approved ( ceua number l-010/04 , ceua number l-002/09 ) protocols . after the indicated times , animals were killed in a co2 chamber , and peritoneal lavage was carried out with 10  ml chilled rpmi 50  ng eotaxin was incubated with 5  g anti - eotaxin neutralizing antibody or 5  g isotype - matched anti - igg2a antibody , in a final volume of 200  l , for 30 minutes , before injection into each balb / c recipient . 4  h later , peritoneal lavage fluid was collected from the injected mice and handled as detailed above . peritoneal lavage cells were washed at 500  g and resuspended in 2  ml rpmi . total counts were carried out in a hemocytometer after a 1  :  10 dilution in turk 's solution . differential counts were done on giemsa - stained ( ice - cold methanol - fixed , air - dried , and giemsa - stained for 5 minutes ) cytocentrifuge smears ( 500  rpm , 8 minutes in a cytospin 3 , thermo scientific , waltham , ma ) , by counting at least 300 cells in 1000x magnification under oil . we used nonpathogenic escherichia coli bacteria ( clone dh5 , provided by dr . z. vasconcelos , from inca and fiocruz , rio de janeiro ) genetically altered to constitutively express the gene for green fluorescent protein ( gfp ) , grown in lb broth . the cells obtained in the peritoneal lavage of balb / c mice , induced by eotaxin or rpmi , were subjected to total cell count as well as differential neutrophil counts as previously described . then 5  10 neutrophils were incubated for 30 minutes , in the dark at room temperature , with the bacteria in a 1  :  400 proportion . the cells were then washed and the resulting cell suspension was run in a facscalibur flow cytometer ( becton dickinson immunocytometry systems , san jose , ca ) , with the acquisition of at least 50.000 events , and analyzed with the help of summit 4.3 software ( dako cytomation , uk ) . 
 for eosinophil transfer studies , where indicated , balb / c , alox or pas mice were sensitized ( 100  g ovalbumin grade iv and 1,6  mg alum in a final volume of 400  l saline per animal , two s. c. injections in the dorsum , at days 0 and 7 ) and challenged ( ovalbumin grade iv , 1  g in 400  l saline i.p . at day 14 ) according to ebihara and colleagues . briefly , bone - marrow cultures were established for 5 days at 37c in 95% air/5% co2 , in rpmi1640 , with 10% fbs and 5  ng / ml il-5 , at a culture density of 1  10 cells / ml . the nonadherent cells were then collected and loaded on top of 3  ml of a histopaque-1083 solution , followed by centrifugation at 400  g , 20c , 35 minutes , without brakes . the mononuclear cell ring and the supernatant were discarded ; the granulocyte - rich pellet was collected , washed and resuspended in 3  ml rpmi , and used for total and differential counts as above . the suspension contained 80% eosinophils , with no neutrophils , and the minor contaminant population consisted of macrophages alone , which do not interfere with the interpretation of transfer experiments . where indicated , naive gata-1 , alox , or pas recipient mice were injected with 1  10 eosinophils from the appropriate donors ( see below ) i.p . , followed by eotaxin 50  ng / ml , and leukocyte accumulation was monitored in the peritoneal lavage fluid 4  h after eotaxin injection , as above . for flow cytometric studies of ccr3 expression , the following modification of this protocol was adopted , for it yielded eosinophils of higher purity : sensitized mice were challenged twice , initially by aerosol exposure ( 1  h , ovalbumin grade ii , 2.5% , w / v , at day 14 ) and 7  h later with soluble ovalbumin i.p . bone marrow was collected 24  h after aerosol challenge and cultured as above , after separation on a percoll gradient ( 75%/60%/45% isotonic percoll , 100  g , 20  min , room temperature ) . the hematopoietic cells from the 45%/60% interface   were cultured at a lower il-5 concentration ( 2.5  ng / ml ) for twice as long ( 10 days ) , yielding a population containing at least 95% eosinophils , with mature morphology . everston , il , usa ) , using the two - tailed t - test for pairwise comparisons . where indicated , anova was also used for multiple comparisons , with the tukey hsd correction and the bonferroni correction for groups of equal and unequal size , respectively . injection of eotaxin in various doses would recruit eosinophils in a relatively short period ( 4  h ) and whether eosinophil accumulation would be selective , as previously observed in sensitized mice , or accompanied by migration of other leukocyte populations . as shown in figure 1(a ) , leukocytes accumulated in response to 50 and 100  ng / cavity eotaxin , in amounts that were significantly different from the rpmi controls ( 0  ng / cavity ) as well as from lower doses of eotaxin ( 10 and 25  ng / cavity ) . these leukocytes included variable numbers of eosinophils ( figure 1(b ) ) , monocytes / macrophages [ 39 , 40 ] ( figure 1(c ) ) , and neutrophils ( figure 1(d ) ) . the morphology of all three leukocyte populations was recognizable without ambiguity , as shown in a representative photomicrograph ( supplementary figure 1 available online at http://dx.doi.org/10.1155/2014/102160 ) . lymphocyte and basophil migration was not significant in any of these doses ( not shown ) . importantly , neutrophils and macrophages greatly outnumbered eosinophils , with counts , respectively , 8.2- and 9.9-fold greater in the experiment shown . for all three leukocyte populations , the dose - response relationships were identical , and in subsequent experiments 50  ng / ml was used as the standard stimulus , since no improvement was observed at a higher dose . despite the heterogeneity of the recruited leukocyte population , neutralization of eotaxin with specific monoclonal antibody brought leukocyte accumulation to negative control levels ( figure 2 ; compare with figure 1(a ) for the 025  ng eotaxin dose range ) , while control antibody of the same isotype with irrelevant specificity had no effect . this confirms that the stimulus for recruitment of all three leukocyte populations is eotaxin itself , not any unidentified contaminant , which by definition would not be neutralized by specific antibody . the kinetics of recruitment of this mixed leukocyte population by eotaxin in naive balb / c mice shows significant accumulation as early as 2  h , with a maximum at 4  h , thereafter decreasing but remaining significant at 12 and 24  h ( figure 3(a ) ) . we can observe very early arrival of eosinophils ( significant from 2  h and remaining so at 12 and 24  h , figure 3(b ) ) . by contrast , accumulation of both monocytes / macrophages ( figure 3(c ) ) and neutrophils ( figure 3(d ) ) became significant only at 4  h. significant accumulation was also observed at 12 and 24  h for monocytes / macrophages and 12  h for neutrophils . eosinophils outlasted neutrophils , but not monocytes / macrophages , in the observation period . for subsequent experiments , the 4  h observation time was chosen , because it showed significant accumulation of eosinophils , monocytes / macrophages , and neutrophils in naive balb / c mice . we first evaluated the effect of eotaxin in naive balb / c mice pretreated with flap inhibitor mk886 or vehicle . balb / c mice responded to eotaxin with significant monocyte / macrophage accumulation by 4  h , which was abolished by mk886 ( figure 4(c ) ) . mk886-pretreated balb / c mice showed no neutrophil migration in response to eotaxin , while migration was significant in vehicle - treated controls ( figure 4(d ) ) . next , we evaluated the effect of eotaxin in nave alox mice , which lack 5-lo , and wild - type pas controls . in alox mice , eotaxin had no significant effect on total leukocyte numbers . by contrast , significant recruitment was observed in pas controls ( figure 5(a ) ) . importantly , alox mice , unlike pas controls , showed no significant eosinophil recruitment ( figure 5(b ) ) . in this genetic background , unlike balb / c , no significant monocyte / macrophage recruitment by eotaxin was observed at this time point ( 4  h ; figure 5(c ) ) , regardless of whether mice were 5-lo - deficient or wild - type ; furthermore , monocyte / macrophage numbers were higher in alox than in pas mice . by contrast , neutrophil recruitment was significant in pas controls and inhibited by 55% in alox mice , although residual neutrophil recruitment remained significant ( figure 5(d ) ) . together , these observations show that , in this genetic background , eosinophils and neutrophils differ in their requirements for 5-lo to migrate in response to eotaxin , which are total for the former but only partial for the latter . the kinetics of mixed leukocyte recruitment in naive balb / c mice raised the issue of whether eotaxin - stimulated eosinophils recruit other leukocyte types . if so , neutrophil and/or monocyte / macrophage migration in response to eotaxin would be decreased in the absence of eosinophils . since naive mice carrying a mutation in the high - affinity gata-1 binding site of the promoter from the gene coding for the gata-1 transcription factor lack eosinophils , we evaluated the effect of eotaxin on leukocyte numbers 4  h after i.p . injection in gata-1 mutant mice and balb / c wild - type controls . in gata-1 mice , unlike balb / c controls , leukocyte numbers in the peritoneal cavity were not significantly increased by eotaxin ( figure 6(a ) ) . as expected , eosinophils were undetectable in gata-1 mice , and effectively recruited by eotaxin in balb / c controls ( figure 6(b ) ) . in both rpmi - treated and eotaxin - treated gata-1 mice , monocyte / macrophages ( which were the predominant resident leukocyte population ) were about twice as numerous as in rpmi - treated balb / c controls ( figure 6(c ) ) , reaching counts comparable to those in eotaxin - treated balb / c . importantly , neutrophil numbers were not significantly increased by eotaxin ( figure 6(d ) ) in gata-1 mice , unlike balb / c controls , suggesting that neutrophil recruitment by eotaxin is eosinophil - dependent . to rule out the possibility that neutrophil migration is somehow defective in this strain , separate control gata-1 mice were injected with thioglycollate broth , which induces an intense neutrophil accumulation in a 4  h period . gata-1 and balb / c mice responded equally well to thioglycollate ( not shown ) , indicating that failure of neutrophil recruitment in gata-1 mice is a feature of their eotaxin response , not evidence of a general defect in neutrophil migration . we further explored this issue by reconstituting a peritoneal eosinophil population in gata-1 mice by transfer of purified ( 90% ) balb / c eosinophils , devoid of neutrophil contamination . total leukocyte counts were not significantly different between gata-1 mice given eotaxin alone , eosinophils alone , or eotaxin plus eosinophils ( figure 6(e ) ) , and this was closely paralleled by monocyte / macrophage counts , which account for most leukocytes in all groups ( figure 6(f ) ) . as expected , eosinophils could be recovered from gata-1 recipients of eosinophils , and eotaxin did not significantly increase their numbers , as the recipients produce no eosinophils of their own ( figure 6(g ) ) . importantly , neutrophil numbers were significantly increased by eosinophil transfer and further significantly increased by the association of eosinophil transfer and eotaxin ( figure 6(h ) ) . together , these data suggest that in naive mice eosinophils mediate the accumulation of neutrophils induced by eotaxin . if , as suggested by the preceding results , neutrophils and monocyte / macrophages accumulate in gata-1 mice as a result of eosinophil activation , not of direct exposure to eotaxin , one should expect the leukocytes harvested from the peritoneal cavity of gata-1 mice to show little or no expression of ccr3 , unlike eosinophils . we have therefore compared the expression of ccr3 in peritoneal lavage leukocytes from balb / c and gata-1 mice collected 4  h after eotaxin injection ( figure 6(i ) ) . mean fluorescence intensity was monitored in the granulocyte region , since our transfer protocol reconstitutes migration of neutrophils , not monocytes / macrophages ( see above ) . no eotaxin - induced recruitment of ccr3 + granulocytes was observed in gata-1 mice ( dotted line ) , unlike balb / c mice ( thin line ) . to make sure that ccr3 + cells would be detectable , if present in a suspension of gata-1 granulocytes , we also added purified balb / c eosinophils to gata-1 leukocytes as a control ( figure 6(i ) , thick line ) . we took advantage of the effectiveness of eosinophil transfer to examine the relationship of 5-lo to the migration of eosinophils , as well as to the secondary recruitment of neutrophils and monocytes / macrophages . a mixed leukocyte population accumulated in the peritoneal cavity of alox recipients of pas eosinophils ( figure 7(a ) ) , 4  h following administration of eotaxin . no significant improvement was observed in pas recipients of pas eosinophils in the same conditions , showing that recruitment is as effective in the alox recipients as in the wild - type recipients . the recruited leukocyte population from alox recipients included eosinophils ( figure 7(b ) ) , comprising both the transferred eosinophils and those recruited by eotaxin administration to the recipients , again reaching levels comparable to those of pas recipients of pas eosinophils . secondary recruitment was observed for both macrophages ( figure 7(c ) ) and neutrophils ( figure 7(d ) ) , with similar effectiveness in comparison to the pas into pas transfers . we next examined whether the critical step requiring 5-lo in this model is the initial eosinophil accumulation , rather than the secondary recruitment of neutrophils by eosinophils . if so , one would predict that direct transfer of alox eosinophils into eosinophil - deficient gata-1 recipients should restore neutrophil accumulation in response to eotaxin . when purified eosinophils from alox bone - marrow cultures were transferred to gata-1 recipients ( figure 7(e ) ) , this rules out the possibility that the step critically dependent on 5-lo is the generation by eosinophils of a neutrophil chemoattractant . on the other hand , as shown above for balb / c eosinophil transfer into gata-1 recipients , monocytes / macrophages were not increased by alox eosinophil transfer at this time point . we further examined whether eosinophil - mediated responses to eotaxin in this model had an effect on the ability of the secondarily recruited neutrophils and their bacterial targets . to do so , mixed leukocyte populations induced by eotaxin ( rpmi in controls ) were collected from naive balb / c mice at 4  h after injection , counted , and mixed for 30 minutes with gfp - expressing e. coli at a bacteria / leukocyte ratio adjusted to 400  :  1 , before analysis by flow cytometry . cells gated in the granulocyte region on the basis of size and complexity were examined for green fluorescence , resulting from both binding and internalization of bacteria . figure 8(a ) shows that eotaxin - stimulated granulocytes bind / internalize fluorescent e. coli bacteria more effectively than those collected from rpmi - injected control mice . this increase in effectiveness is detectable as an increased fraction of granulocytes binding bacteria ( figure 8(b ) ) and an increased mean fluorescent intensity ( figure 8(c ) ) . this suggests that eosinophil - mediated recruitment of neutrophils is accompanied by an increased capacity to bind and/or ingest bacteria . we describe here a mixed leukocyte accumulation occurring in the peritoneal cavity of naive mice injected with eotaxin . this is , to our knowledge , the first experimental evidence that recruitment of neutrophils and macrophages by eotaxin in nonsensitized animals is mediated by eosinophils . for neutrophils , recruitment was associated with an increased ability to bind / ingest bacteria and therefore might have an impact on antimicrobial defenses in specific conditions . because the ability of eosinophils to act as effective antimicrobial defenses is limited by their scarcity , these findings also highlight conditions in which , by recruiting much larger numbers of cells with well - characterized microbicidal function , eosinophils actually overcome this theoretical disadvantage . we will below address a number of specific points which are important for putting our observations in a proper perspective . migration of all three leukocyte types in balb / c mice was induced by eotaxin , as shown by identical dose - response relationships and overlapping kinetics , as well as by identical effects of neutralizing eotaxin with specific antibodies . the relationship of this migration to the expression of ccr3 , by contrast , is more complex . lymphocytes , some of which have been shown by others to express ccr3 [ 41 , 42 ] , were not attracted by eotaxin to the peritoneal cavity of naive mice in significant numbers . on the other hand , despite the commonly held view that ccr3 expression is restricted to eosinophils [ 4 , 18 , 20 , 22 , 23 ] , basophils [ 24 , 25 ] , eosinophil and basophil progenitors / precursors [ 26 , 27 ] , t cell subsets [ 41 , 42 ] , and smooth muscle cells , several studies have suggested that human and murine neutrophils and macrophages can also express ccr3 , at least in specific experimental settings [ 28 , 32 ] , as suggested by studies in neutrophils . this would imply that all three leukocyte populations shown to be recruited in our study in wild - type ( balb / c , pas ) mice could be simply responding to eotaxin binding to ccr3 at the individual cell level , with no contribution from cellular interactions involving eosinophils . if so , there should be no decrease in neutrophil or macrophage accumulation by eliminating eosinophils , but one should expect neutrophils and macrophages to express ccr3 at significant levels even in eosinophil - deficient gata-1 mice . this possibility , however , has been directly ruled out by the demonstration that eotaxin in gata-1 mutant mice does not recruit neutrophils nor macrophages . the evidence for cellular interactions in the neutrophil response to eotaxin is reinforced by experiments using the same strain , which show neutrophil recruitment following transfer of highly purified balb / c eosinophils . finally , we observed no significant accumulation of ccr3 + granulocytes in eotaxin - injected gata-1 mice . by contrast , gata-1 mice had constitutively increased macrophage numbers in the peritoneal cavity , which were unaffected by 4  h of eotaxin administration , both with and without eosinophil transfer . it is possible that the gata-1 mutation affects the cellular function , tissue distribution , and/or turnover of monocytes / macrophages so as to prevent responses to eotaxin , regardless of whether these are mediated or not by eosinophils . therefore , we can not conclude from our present observations in gata-1 mice alone that eosinophils also recruit monocytes / macrophages . direct evidence for eosinophil recruitment of monocytes / macrophages was , however , obtained through transfer of eosinophils from pas donors into alox mice . importantly , in the absence of eosinophil transfer , alox mice showed neither eosinophil nor monocyte / macrophage recruitment by eotaxin . interestingly , although in the direct stimulation protocol alox mice resembled gata-1 mice , in their absence of monocyte / macrophage accumulation by 4  h , it is likely that different mechanisms underlie these similar outcomes , since ( a ) a similar failure to respond to eotaxin with monocyte / macrophage accumulation was observed in wild - type pas controls and can not therefore be ascribed to the absence of active 5-lo ; ( b ) transfer experiments show that eosinophil transfer from pas donors allows a significant recruitment of monocytes / macrophages by eotaxin in alox recipients . these observations suggest that an active 5-lo is not required for monocytes / macrophages ( or neutrophils ) to respond to eotaxin , provided eosinophils are present . overall , the data indicate that eotaxin recruits a mixed leukocyte population in naive mice through a mechanism dependent on eosinophils . evidence that eosinophils play an active role is provided by the observation that full effect in eosinophil transfer experiments requires both eosinophils and eotaxin , which would not be expected if eosinophils played a merely passive or permissive role . on the other hand , in transfer experiments about 10% of the transferred eosinophils were recovered by 4  h eotaxin stimulation of the recipients . this raises the issue of whether the remaining transferred eosinophils underwent changes such as degranulation   or release of extracellular traps , which might represent a significant difference relative to the direct ( nontransfer ) protocol used in the initial experiments . mixed leukocyte recruitment by eotaxin in naive mice shows the same dependence on 5-lo that was observed for selective eosinophil recruitment in sensitized mice . hence , it is likely that eosinophil accumulation itself , the shared feature in both models , is the 5-lo - dependent step . this is consistent with the observation that alox eosinophils , when directly transplanted to the peritoneal cavity of gata-1 recipients ( which are unable to respond to eotaxin by accumulation of neutrophils ) , are able to mediate the neutrophil recruitment induced by eotaxin . importantly , further recruitment of eosinophils occurs in eotaxin - stimulated alox recipients of pas eosinophils , where the only cells bearing a functional 5-lo are the transferred eosinophils . this suggests that eosinophils can be a source as well as a target for a 5-lo pathway product , such as ltb4 . ltb4 was previously shown to selectively attract eosinophils , in a model in which eotaxin duplicated the effect of antigen in a 5-lo - dependent manner . furthermore , there is significant evidence that recruitment involves interactions between cytokines and lipid mediators . in neutrophil migration , ltb4 represents a signaling relay , raising the possibility that it acts similarly in eosinophils . whatever mechanism is involved , eosinophil generation of a 5-lo - derived neutrophil chemoattractant is not required for the eosinophil - dependent secondary recruitment of neutrophils in eotaxin - injected nave mice . while in previous studies of sensitized mice , ltb4 , as did antigen and eotaxin , selectively recruited eosinophils , it remains to be determined whether it accounts for the rapid eosinophil recruitment to the peritoneal cavity of the eotaxin - injected nonsensitized mice in the present study . a related issue for further investigation is whether 5-lo is required for the increased effectiveness of bacterial binding that was detected in balb / c leukocytes , as ltb4 is known to activate as well as attract neutrophils . despite a common requirement for 5-lo , leukocyte recruitment by eotaxin differs in several aspects between naive versus sensitized mice , especially the lack of eosinophil selectivity in the former , as opposed to the latter . in sensitized mice , selective eosinophil recruitment was observed with widely different chemical stimuli ( allergen , eotaxin , or ltb4 ) . it is unlikely , therefore , that such selectivity reflects some features of eotaxin signaling and even less of ltb4 signaling ( which should be very effective in mice having normal neutrophil numbers ) . alternatively , the failure of eotaxin and ltb4 to recruit neutrophils and monocytes / macrophages in sensitized mice could involve changes in the expression of adhesion proteins at endothelial surfaces , which would prevent their emigration from blood vessels to peritoneal cavity , regardless of whether the chemoattractant is ltb4 or eotaxin . we have not examined this possibility , since our current observations , which are centered on responses from nonsensitized animals , do not depend on clarifying mechanisms that were not applicable to the present conditions . we view our findings as manifestations of innate immunity , because of ( a ) the very fast kinetics of eosinophil and neutrophil accumulation ; ( b ) the recruitment of neutrophils and macrophages in the absence of significant lymphocyte accumulation ; ( c ) the detectable increase in granulocyte binding of live extracellular bacteria in the absence of antibodies . on the other hand , the fast recruitment of monocytes / macrophages by eotaxin - exposed eosinophils raises the issue of whether eosinophils could also enhance protection from more specialized pathogens , such as the intracellular mycobacteria and protozoa that cause chronic infections , which are usually handled by monocytes / macrophages . relatively little attention has been paid to the possibility that eosinophils play a role in fighting microbial pathogens with the help of other leukocyte types . our observations suggest that small numbers of eosinophils might recruit a large neutrophil and/or macrophage infiltrate . while this would make eosinophils surprisingly effective players in innate immunity , this might paradoxically obscure their contribution , if their contribution were to be taken as commensurate with their numbers in inflammatory infiltrates , where they would often amount to no more than one - tenth of total leukocytes . it is therefore fortunate that , in transfer experiments of wild - type and mutant eosinophils into eosinophil - null gata-1 mice , eosinophil recruitment of neutrophils can be unequivocally demonstrated . in view of the differences between naive and sensitized models in this respect , it is of interest to determine whether this eosinophil functional capacity is modified by allergen sensitization of the host and whether such a change in innate immune functions can be duplicated by passively or actively sensitizing the host . several , but not all , of the observations reported here are consistent with those of previous studies , carried out by other groups in different experimental models . eotaxin recruitment of a mixed leukocyte population , including neutrophils and macrophages , was described in human subjects ; das and colleagues   reported that eotaxin was effective when injected in the peritoneal cavity of mice but not in a dorsal air pouch , drawing attention to the important differences between challenge sites responding to the same chemically defined stimulus . responses in the air pouch occurred after local inoculation of mast cell - containing peritoneal cell populations , but allergen sensitization was essential to local responses to eotaxin in this transfer model . harris and colleagues   confirmed that mast cells were important for full responses to eotaxin and further showed that eotaxin responses were blocked by 5-lo inhibitors . together , these studies suggest that eotaxin effectiveness is constrained in vivo by several factors that may be absent from in vitro ( e. g. , migration chamber or flow cytometric ) studies . none of these published studies , however , evaluated the contribution of the recruited eosinophils themselves . we suggest that a cytokine , rather than a 5-lo derivative , is released by eosinophils in the peritoneal cavity , once they have been recruited by eotaxin in the presence of an active 5-lo , or , alternatively , directly inoculated in the cavity through a transfer protocol . one hypothesis that could reconcile our observations with those of das and harris and their colleagues [ 46 , 47 ] would involve the amplification of the role of eosinophils through interactions with resident peritoneal mast cells , since mast cells are an important source of neutrophil chemoattractants , including tnf- .
the roles of eosinophils in antimicrobial defense remain incompletely understood . in ovalbumin - sensitized mice , 
 eosinophils are selectively recruited to the peritoneal cavity by antigen , eotaxin , or leukotriene(lt)b4 , a 5-lipoxygenase ( 5-lo ) metabolite . 
 5-lo blockade prevents responses to both antigen and eotaxin . 
 we examined responses to eotaxin in the absence of sensitization and their dependence on 5-lo . 
 balb / c or pas mice and their mutants ( 5-lo - deficient alox ; eosinophil - deficient gata-1 ) were injected i.p . with eotaxin , eosinophils , or both , and leukocyte accumulation 
 was quantified up to 24  h. significant recruitment of eosinophils by eotaxin in balb / c , up to 24  h , was accompanied by much larger numbers of recruited neutrophils and monocytes / macrophages . 
 these effects were abolished by eotaxin neutralization and 5-lo - activating protein inhibitor mk886 . in alox ( but not pas ) mice , eotaxin recruitment was abolished for eosinophils and halved for neutrophils . in gata-1 mutants , 
 eotaxin recruited neither neutrophils nor macrophages . 
 transfer of eosinophils cultured from bone - marrow of balb / c donors , or from alox donors , into gata-1 mutant recipients , i.p . 
 , restored eotaxin recruitment of neutrophils and showed that the critical step dependent on 5-lo is the initial recruitment of eosinophils by eotaxin , not the secondary neutrophil accumulation . 
 eosinophil - dependent recruitment of neutrophils in naive balb / c mice was associated with increased binding of bacteria .