BACKGROUND
the view that the central nervous system  is an immunologically privileged site has been widely accepted based on classic experiments such as those of medawar  <cit> , who showed that rabbit skin allografted into the cns failed rejection.
this immunologic privilege is specifically related to the low levels of mhc class ii expression  <cit> , which is responsible for antigen presentation in the brain.
an additional mechanism for maintaining immune privilege is the presence of the blood-brain barrier , which is a semi-permeable barrier, composed of endothelial cells that restrict the interface between the brain and immune circulatory products.
specific regions of the brain, the circumventricular organs, lie outside the bbb  <cit>  and are therefore subject to the effects of serum-derived substances including immune factors.
one of those regions is the pineal gland, which has been studied extensively in recent years for its immuno-regulatory function  <cit> .
the gland secretes a potent hormone, melatonin, that tends to stimulate the immune system and its production of cytokines  <cit> .
because the pineal gland's neuroendocrine functions are closely linked to the 24-hour light:dark cycle  <cit> , these pineal-immune interactions are also thought to have a temporal component.
evidence for effects of the pineal gland on photoperiodic changes in the immune system has been collected in both birds and mammals  <cit> .
interactions between the neuroendocrine and immune systems presume that feedback mechanisms are present from one system to the other via immune soluble factors.
antigenic stimulation  <cit> , inflammation  <cit>  and treatment with cytokines  <cit>  have all been shown to modulate the neuroendocrine functions of the gland, although the cellular and molecular mechanisms of this feedback are still poorly understood.
one cell population in the pineal gland that has been implicated as mediators of these immune effects on pinealocyte functions are the microglia/macrophages.
these cells regulate pinealocyte neurite length and serotonin content in an invitro system and they upregulate cytokine expression, mhc class ii and other surface antigens in response to cytokines and bacterial wall components  <cit> .
feedback of the immune system on the pineal gland is further indicated by reports of accumulations of lymphocytes in the pineal of both avian and mammalian species  <cit> .
in some species such as the chicken, these accumulations of lymphocytes account for up to 30% of the total volume of the gland  <cit> .
the large size of this pineal-associated lymphoid tissue  and the extent of lymphocyte infiltration suggest novel mechanisms of neuro-immune interactions in this part of the brain.
the presence of palt raises several important questions regarding the mechanisms of interactions between lymphocytes and the pineal gland, including effects on circadian rhythms in both the immune system and the pineal gland.
to date, there are no studies on possible changes in the composition and distribution of lymphocytes in the pineal over a light: dark cycle.
accordingly, the purpose of this study was to further characterize the phenotypes and distribution of lymphocytes in the chicken pineal gland, and to determine if lymphocyte subsets vary over the 24-h light: dark cycle.
RESULTS
general histology of the pineal gland
the pineal gland in the chicken develops through the formation of a series of vesicles or follicles separated by prominent septa  as previously described  <cit> .
each follicle has a central lumen into which the apical ends of the pinealocytes protrude.
the interstitial spaces contain connective tissue, smaller capillaries and mononuclear  cells, which occurred either singly or in small clusters.
palt was typically located along the periphery of the gland, but could be found centrally betweem adjacent follicles.
for descriptive purposes, palt refers to the larger accumulations of mononuclear cells with distinct borders separating it from the pineal tissue.
most of the cells within palt resembled small lymphocytes, although larger, pleomorphic cells were also present .
these mononuclear cells were also frequently seen infiltrating the pineal follicle .
mitotic figures were commonly observed throughout the palt , inter-follicular spaces and even within the pineal follicles.
lymphocyte phenotypes and distributions
immunohistochemical phenotyping of lymphocytes within the palt and pineal tissue showed that the majority of lymphocytes stained positively for cd <dig> .
cd3+ cells were also commonly seen within the pineal inter-follicular septa bordering palt or within the gland at some distance from the palt.
two-color immuno-staining revealed that the majority of cd3+ lymphocytes were also cd4+ .
fewer cd3+ lymphocytes in the palt co-localized with cd8α.
these cd3+cd8α+ cells tended to distribute randomly throughout the palt, although on occasion were seen localized along the periphery of the palt.
antibodies to both tcr <dig>  and tcr <dig>  showed staining that co-localized with cd3-positive lymphocytes, although the frequency of tcrγδ+ cells was significantly less than the frequency of tcrαβ/vβ <dig> + cells.
the tcrγδ+ cells tended to be found along the periphery of the palt.
bu-1+ cells were present within palt and did not co-localize with cd <dig> .
overall, there were few bu-1+ cells compared to cd3+ cells.
similar to the γδ-positive cells, bu-1+ cells were found typically along the periphery of the palt.
on two occasions, clusters of bu-1+ cells surrounded by cd3+ cells were seen within the palt.
staining with antibodies against igm and igλ  revealed positive cells, with denser staining from igλ.
however, there was also extensive labeling within the lumen of follicles and the interstitial spaces, possibly due to soluble antibodies.
this background staining negated the effectiveness of the anti-igm and anti-igλ antibodies for use in image analysis of lymphocyte phenotypes.
bu-1+ cells resembling microglia/macrophages were also seen within the pineal follicles .
the microglia/macrophages were easily distinguished by their ramified morphologies.
in contrast to the common occurrence of t-cell lymphocyte phenotypes in the interstitial spaces between follicles, round, mononuclear bu-1+ cells were rarely seen in these compartments.
large numbers of t-lymphocytes were found within the interfollicular spaces occurring either singly or in small clusters.
the t-cells were also observed within the pineal follicles, even bordering the central lumen .
the frequency of cd4+ cells approximated the frequency of cd4-/cd8+ cells within the interfollicular spaces.
image analysis of lymphocyte phenotypes
measurements of the profile areas of the palt and pineal tissue showed that palt made up approximately 7–12% of the total pineal area.
the average profile area of palt in glands collected at the light: dark interphase was approximately  <dig>  μm <dig> whereas the glands collected at the dark: light interphase was approximately  <dig>  μm <dig>  in contrast, the average profile area of pineal tissue did not change between the two time points studied .
image analysis of lymphocyte phenotypes in palt revealed that approximately 80% of positively stained mononuclear cells were cd3+ t cells with the remaining 20–25% comprising bu-1+ cells .
estimates of the number of cells per unit area confirmed that the majority of cd3+ cells were cd4+  and αβ+  when all glands were combined.
cd8α+ cells accounted for approximately 20% of the cd3+ cells.
the tcrγδ+ cells had the lowest frequency .
the only phenotype that showed a significant difference between the two time points studied were the tcrαβ/vβ <dig> + cells, which exhibited an 18% decline  at the light: dark interphase .
each of the phenotypes of t-lymphocytes was observed in the interstitial spaces as well as within the pineal follicles.
as noted above, bu-1+ cells were only rarely seen in the interstitial spaces, but bu-1+ microglia/macrophages were common in the pineal follicles.
in contrast to the palt, where the ratio of cd4+/cd8α+ cells was about 4: <dig> , the ratio of cd4+/cd8α+ cells in the non-palt compartments was approximately 1: <dig> .
similarly, there was approximately a 4-fold difference in the ratio of tcrαβ/vβ1+/tcrγδ+ cells for palt compared to the non-palt regions of the gland .
there were no significant differences in the numerical density of lymphocyte phenotypes between the two time points studied in the non-palt regions, but it is noteworthy that the density of tcrαβ/vβ1+ cells increased 40% at the light: dark interphase , which was opposite to the trend for this phenotype in the palt.
bu-1-positive cells were common in the pineal tissue, although b cells could not be readily distinguished from the microglia/macrophages, which are also stained with this antibody.
immuno-staining with anti-igm and anti-igλ  did not resolve the b cells because these antibodies stained secretory products throughout the pineal tissue, including the lumen of follicles.
because igλ labels most of the immunoglobulin fraction , it was observed to stain more intensely than igm.
the day: night differences in the area density  of the bu-1+ microglia/macrophages within pineal tissue approached significance as analyzed by a two-tailed student's t-test  with a 43% decline at the light: dark interphase  compared to the dark: light interphase .
discussion
the histology of the palt in this study was in general agreement with previous reports  <cit> .
moreover, the present finding that palt occupied approximately 10% of the total pineal area in 14– <dig> day old chicks was reasonably close to cogburn's  <cit>  estimate that palt occupied 18% of the total volume in older white leghorn chickens.
the phenotypes and distributions of lymphocytes in palt resembled the gut-associated lymphoid tissues  found in mucosal structures such as the proventriculus  <cit> .
palt and galt each showed a higher concentration of t cells centrally , whereas tcrγδ+ and bu-1+ cells were found more peripherally.
our results are consistent with the hypothesis that lymphocyte-pineal interactions vary over the light:dark cycle.
the 2-fold change in profile area of palt and the decline in density of tcrαβ/vβ <dig> + cells at the light:dark interphase suggest that lymphocyte trafficking in the palt varies with time.
however, additional samples are required over the 24-h light:dark cycle as well as other criteria  need to be met before a circadian rhythm in pineal lymphocyte trafficking can be definitively established in this system.
because immunohistochemistry allows analysis of only a small percentage of the tissue in any gland, we are undertaking to develop methodologies of fluorescence activated cell sorting  to analyze lymphocyte subsets in the gland.
cogburn and glick  <cit>  concluded that palt formation is dependent on the bursa and thymus based on the reduction in size of palt following bursectomy and/or thymectomy with whole body irradiation.
in a later study, cogburn and glick  <cit>  showed that tritium-labelled lymphocytes from the thymus and bursa migrated to the pineal gland, but only in older  chicks.
studies are currently underway to determine whether soluble chemotactic factors from chicken pineal glands induce migration of specific lymphocyte subtypes using an invitro migration assay.
this specificity of migration is suggested by the present study showing that the ratios of cd8+ to cd4+, and tcrγδ+ to tcrαβ/vβ <dig> + cells are nearly four fold greater in the pineal tissue compared to that found in palt.
the close correlation of cd <dig> to tcrγδ is supported by chen et al.
<cit>  who reported that the majority of tcrγδ cells expressed cd <dig>  a preferential recruitment of cd8+ and tcrγδ+ cells into the pineal tissue was also found to occur in the retina  <cit> , which is notable considering that the pineal gland shares homologies related to common photoreceptive functions  <cit> .
imagawa et al.
<cit>  speculated that the lymphocytes played a role in the normal surveillance of the retina and functioned to remove cell debris as well as remaining in the retina as memory t cells.
the scarcity of bu-1+ cells in the inter-follicular  compartment is further evidence of the preferential recruitment of lymphocytes to the pineal tissue.
image analysis of these cells revealed that they comprised 20–25% of palt lymphocytes, similar to that found in the spleen  <cit> .
cogburn and glick  <cit>  described a much higher proportion of b lymphocytes  based on a cytotoxic assay in older  chicks of a different strain .
however, olah and magyar  <cit> , using the bu-1b antibody in the new hapmpshire strain showed staining patterns that were similar to those observed in the present study.
as reported here, the bu-1-positive cells were predominantly found along the periphery of palt and the authors also described large clusters of bu-1-positive cells, which they described as germinal centers  <cit> .
lymphocyte interactions with pinealocytes are clearly implicated by their common occurrence within the pineal follicles, which in some cases exhibited heavy infiltration  <cit> , even as far as the central lumen of the pineal follicles .
the proliferation of these cells in the non-palt pineal compartments is greater than in the palt based on tritiated-thymidine incorporation studies  <cit>  and counts of mitotic figures .
the enhanced proliferation of lymphocytes in the pineal tissue  suggests that the pineal cells produce blastogenic products that may also affect the differentiation of the lymphocytes  <cit> .
these results are additional evidence that lymphocytes traffic regularly between palt and the pineal follicles.
the bu-1+ cells observed within the pineal follicles are most likely microglia/macrophages because their distribution and ramified morphology resemble the microglia/macrophages described in the mammalian pineal gland  <cit> .
to our knowledge, this is the first description of these cells in the avian pineal gland and, as such, represent ideal candidates as cellular interfaces between the immune system and the neuroendocrine pinealocytes.
in contrast to most other parts of the brain, microglia/macrophages in the pineal gland are constitutively active as evidenced by the expression of mhc class ii  <cit> .
because these cells are actively involved in antigen presentation, and secrete potent cytokines , they have been implicated in the regulation of pinealocyte differentiation and neuroendocrine functions.
tsai and mcnulty  <cit>  demonstrated in the rat that pineal microglia regulate pinealocyte neurite length and the production of serotonin in vitro.
the production of melatonin could also be stimulated in vivo by treatment with il-1β  <cit> , presumably produced by these activated microglia/macrophages.
more recently, jiang-shieh et al.
<cit>  demonstrated that pineal microglia/macrophages are in constant surveillance of blood-borne pathogens and presumably other serum-derived substances.
day-night differences in the expression of the bu- <dig> epitope in microglia/macrophages are consistent with their hypothesized role as interfaces in the interaction with palt lymphocytes.
increased expression of bu- <dig> occurred at the dark:light interphase , the time immediately following when cells were exposed to the highest levels of melatonin, which is produced during the dark phase .
the time-point corresponded to the rise in area density of tcrαβ/vβ <dig> + cells in the palt.
this temporal relationship may be explained by increased secretion of microglial cytokines and chemokines assuming that increased expression of bu- <dig> represents activation of the microglia.
the terminology used to describe microglia in the pineal gland is complicated by the fact that this organ is outside the blood-brain barrier.
although perry and gordon  <cit>  described similar populations of cells in the choroid plexus and meninges as macrophages, we include the term "microglia" because the pineal gland is part of the brain, a terminology adapted by other authors  <cit> .
however, it is not clear whether these cells are undifferentiated microglia or resident macrophages derived from circulating monocytes.
in addition to b-cells, the bu- <dig> antibody stains cells of the monocyte line and recognizes a highly glycosylated transmembrane protein that has no similarity to known mammalian markers  <cit> .
the bu- <dig> antibody has also been shown to stain microglia within the sensory epithelium of the ear  <cit> .
the functional significance of lymphocyte trafficking in the pineal gland has yet to be determined, although there are two overall working hypotheses, which are not mutually exclusive.
one hypothesis addresses the mechanisms involved in the recruitment of lymphocytes to the gland and their possible differentiation to form distinct subpopulation, which are involved in immunological surveillance of the cns including the cerebrospinal fluid .
in many species, the gland and the palt are juxtaposed to the choroid plexus, which produces csf.
lymphocytes have been shown to migrate through the choroid plexus and enter the csf at these locations  <cit> .
the second hypothesis addresses immunological mechanisms regulating the neuroendocrine functions of the gland.
as noted above, several studies have shown that immune-derived cytokines modulate both neural and neuroendocrine activities.
it is therefore conceivable that lymphocyte trafficking in the pineal regulates the circadian production of melatonin.
this potential circuit could represent a direct feedback of the immune system on a gland that is known to regulate immune functions.
CONCLUSIONS
in conclusion, this study has provided a detailed quantitative description of pineal-associated lymphocyte phenotypes at two time points over the 24-hour light:dark cycle.
our results show that lymphocytes comprise a significant component of pineal tissue in this species.
the data further suggest that cd8+/ tcrγδ+ cells are preferentially recruited to the pinealocytes and that pineal-lymphocyte interactions are dynamically linked to the light:dark cycle.
pineal microglia/macrophages are proposed to play an important role in these interactions through their secretion of cytokines and chemokines.
the unique accumulation of lymphocytes in this part of the brain has important functional implications with regard to homeostatic mechanisms of neuro-immune interactions, especially those involving circadian components of both the immune and nervous systems and the possible role of these cells in immune surveillance in other parts of the cns.
further studies of the cellular and molecular mechanisms of these homeostataic pineal-lymphocyte interactions will provide a better understanding of disease processes in the nervous system.

 the view that the central nervous system is an immunologically privileged site has been widely accepted based on classic experiments such as those of medawar, who showed that rabbit skin allografted into the cns failed rejection. this immunologic privilege is specifically related to the low levels of mhc class ii expression, which is responsible for antigen presentation in the brain. an additional mechanism for maintaining immune privilege is the presence of the blood-brain barrier, which is a semi-permeable barrier, composed of endothelial cells that restrict the interface between the brain and immune circulatory products. specific regions of the brain, the circumventricular organs, lie outside the bbb and are therefore subject to the effects of serum-derived substances including immune factors. one of those regions is the pineal gland, which has been studied extensively in recent years for its immuno-regulatory function. the gland secretes a potent hormone, melatonin, that tends to stimulate the immune system and its production of cytokines. because the pineal gland's neuroendocrine functions are closely linked to the 24-hour light:dark cycle, these pineal-immune interactions are also thought to have a temporal component. evidence for effects of the pineal gland on photoperiodic changes in the immune system has been collected in both birds and mammals. interactions between the neuroendocrine and immune systems presume that feedback mechanisms are present from one system to the other via immune soluble factors. antigenic stimulation, inflammation and treatment with cytokines have all been shown to modulate the neuroendocrine functions of the gland, although the cellular and molecular mechanisms of this feedback are still poorly understood. one cell population in the pineal gland that has been implicated as mediators of these immune effects on pinealocyte functions are the microglia/macrophages. these cells regulate pinealocyte neurite length and serotonin content in an invitro system and they upregulate cytokine expression, mhc class ii and other surface antigens in response to cytokines and bacterial wall components. feedback of the immune system on the pineal gland is further indicated by reports of accumulations of lymphocytes in the pineal of both avian and mammalian species. in some species such as the chicken, these accumulations of lymphocytes account for up to 30% of the total volume of the gland. the large size of this pineal-associated lymphoid tissue and the extent of lymphocyte infiltration suggest novel mechanisms of neuro-immune interactions in this part of the brain. the presence of palt raises several important questions regarding the mechanisms of interactions between lymphocytes and the pineal gland, including effects on circadian rhythms in both the immune system and the pineal gland. to date, there are no studies on possible changes in the composition and distribution of lymphocytes in the pineal over a light: dark cycle. accordingly, the purpose of this study was to further characterize the phenotypes and distribution of lymphocytes in the chicken pineal gland, and to determine if lymphocyte subsets vary over the 24-h light: dark cycle.  general histology of the pineal gland the pineal gland in the chicken develops through the formation of a series of vesicles or follicles separated by prominent septa as previously described. each follicle has a central lumen into which the apical ends of the pinealocytes protrude. the interstitial spaces contain connective tissue, smaller capillaries and mononuclear cells, which occurred either singly or in small clusters. palt was typically located along the periphery of the gland, but could be found centrally betweem adjacent follicles. for descriptive purposes, palt refers to the larger accumulations of mononuclear cells with distinct borders separating it from the pineal tissue. most of the cells within palt resembled small lymphocytes, although larger, pleomorphic cells were also present. these mononuclear cells were also frequently seen infiltrating the pineal follicle. mitotic figures were commonly observed throughout the palt, inter-follicular spaces and even within the pineal follicles. lymphocyte phenotypes and distributions immunohistochemical phenotyping of lymphocytes within the palt and pineal tissue showed that the majority of lymphocytes stained positively for cd. cd3+ cells were also commonly seen within the pineal inter-follicular septa bordering palt or within the gland at some distance from the palt. two-color immuno-staining revealed that the majority of cd3+ lymphocytes were also cd4+. fewer cd3+ lymphocytes in the palt co-localized with cd8α. these cd3+cd8α+ cells tended to distribute randomly throughout the palt, although on occasion were seen localized along the periphery of the palt. antibodies to both tcr and tcr showed staining that co-localized with cd3-positive lymphocytes, although the frequency of tcrγδ+ cells was significantly less than the frequency of tcrαβ/vβ + cells. the tcrγδ+ cells tended to be found along the periphery of the palt. bu-1+ cells were present within palt and did not co-localize with cd. overall, there were few bu-1+ cells compared to cd3+ cells. similar to the γδ-positive cells, bu-1+ cells were found typically along the periphery of the palt. on two occasions, clusters of bu-1+ cells surrounded by cd3+ cells were seen within the palt. staining with antibodies against igm and igλ revealed positive cells, with denser staining from igλ. however, there was also extensive labeling within the lumen of follicles and the interstitial spaces, possibly due to soluble antibodies. this background staining negated the effectiveness of the anti-igm and anti-igλ antibodies for use in image analysis of lymphocyte phenotypes. bu-1+ cells resembling microglia/macrophages were also seen within the pineal follicles. the microglia/macrophages were easily distinguished by their ramified morphologies. in contrast to the common occurrence of t-cell lymphocyte phenotypes in the interstitial spaces between follicles, round, mononuclear bu-1+ cells were rarely seen in these compartments. large numbers of t-lymphocytes were found within the interfollicular spaces occurring either singly or in small clusters. the t-cells were also observed within the pineal follicles, even bordering the central lumen. the frequency of cd4+ cells approximated the frequency of cd4-/cd8+ cells within the interfollicular spaces. image analysis of lymphocyte phenotypes measurements of the profile areas of the palt and pineal tissue showed that palt made up approximately 7–12% of the total pineal area. the average profile area of palt in glands collected at the light: dark interphase was approximately μm whereas the glands collected at the dark: light interphase was approximately μm in contrast, the average profile area of pineal tissue did not change between the two time points studied. image analysis of lymphocyte phenotypes in palt revealed that approximately 80% of positively stained mononuclear cells were cd3+ t cells with the remaining 20–25% comprising bu-1+ cells. estimates of the number of cells per unit area confirmed that the majority of cd3+ cells were cd4+ and αβ+ when all glands were combined. cd8α+ cells accounted for approximately 20% of the cd3+ cells. the tcrγδ+ cells had the lowest frequency. the only phenotype that showed a significant difference between the two time points studied were the tcrαβ/vβ + cells, which exhibited an 18% decline at the light: dark interphase. each of the phenotypes of t-lymphocytes was observed in the interstitial spaces as well as within the pineal follicles. as noted above, bu-1+ cells were only rarely seen in the interstitial spaces, but bu-1+ microglia/macrophages were common in the pineal follicles. in contrast to the palt, where the ratio of cd4+/cd8α+ cells was about 4:, the ratio of cd4+/cd8α+ cells in the non-palt compartments was approximately 1:. similarly, there was approximately a 4-fold difference in the ratio of tcrαβ/vβ1+/tcrγδ+ cells for palt compared to the non-palt regions of the gland. there were no significant differences in the numerical density of lymphocyte phenotypes between the two time points studied in the non-palt regions, but it is noteworthy that the density of tcrαβ/vβ1+ cells increased 40% at the light: dark interphase, which was opposite to the trend for this phenotype in the palt. bu-1-positive cells were common in the pineal tissue, although b cells could not be readily distinguished from the microglia/macrophages, which are also stained with this antibody. immuno-staining with anti-igm and anti-igλ did not resolve the b cells because these antibodies stained secretory products throughout the pineal tissue, including the lumen of follicles. because igλ labels most of the immunoglobulin fraction, it was observed to stain more intensely than igm. the day: night differences in the area density of the bu-1+ microglia/macrophages within pineal tissue approached significance as analyzed by a two-tailed student's t-test with a 43% decline at the light: dark interphase compared to the dark: light interphase. discussion the histology of the palt in this study was in general agreement with previous reports. moreover, the present finding that palt occupied approximately 10% of the total pineal area in 14– day old chicks was reasonably close to cogburn's estimate that palt occupied 18% of the total volume in older white leghorn chickens. the phenotypes and distributions of lymphocytes in palt resembled the gut-associated lymphoid tissues found in mucosal structures such as the proventriculus. palt and galt each showed a higher concentration of t cells centrally, whereas tcrγδ+ and bu-1+ cells were found more peripherally. our results are consistent with the hypothesis that lymphocyte-pineal interactions vary over the light:dark cycle. the 2-fold change in profile area of palt and the decline in density of tcrαβ/vβ + cells at the light:dark interphase suggest that lymphocyte trafficking in the palt varies with time. however, additional samples are required over the 24-h light:dark cycle as well as other criteria need to be met before a circadian rhythm in pineal lymphocyte trafficking can be definitively established in this system. because immunohistochemistry allows analysis of only a small percentage of the tissue in any gland, we are undertaking to develop methodologies of fluorescence activated cell sorting to analyze lymphocyte subsets in the gland. cogburn and glick concluded that palt formation is dependent on the bursa and thymus based on the reduction in size of palt following bursectomy and/or thymectomy with whole body irradiation. in a later study, cogburn and glick showed that tritium-labelled lymphocytes from the thymus and bursa migrated to the pineal gland, but only in older chicks. studies are currently underway to determine whether soluble chemotactic factors from chicken pineal glands induce migration of specific lymphocyte subtypes using an invitro migration assay. this specificity of migration is suggested by the present study showing that the ratios of cd8+ to cd4+, and tcrγδ+ to tcrαβ/vβ + cells are nearly four fold greater in the pineal tissue compared to that found in palt. the close correlation of cd to tcrγδ is supported by chen et al. who reported that the majority of tcrγδ cells expressed cd a preferential recruitment of cd8+ and tcrγδ+ cells into the pineal tissue was also found to occur in the retina, which is notable considering that the pineal gland shares homologies related to common photoreceptive functions. imagawa et al. speculated that the lymphocytes played a role in the normal surveillance of the retina and functioned to remove cell debris as well as remaining in the retina as memory t cells. the scarcity of bu-1+ cells in the inter-follicular compartment is further evidence of the preferential recruitment of lymphocytes to the pineal tissue. image analysis of these cells revealed that they comprised 20–25% of palt lymphocytes, similar to that found in the spleen. cogburn and glick described a much higher proportion of b lymphocytes based on a cytotoxic assay in older chicks of a different strain. however, olah and magyar, using the bu-1b antibody in the new hapmpshire strain showed staining patterns that were similar to those observed in the present study. as reported here, the bu-1-positive cells were predominantly found along the periphery of palt and the authors also described large clusters of bu-1-positive cells, which they described as germinal centers. lymphocyte interactions with pinealocytes are clearly implicated by their common occurrence within the pineal follicles, which in some cases exhibited heavy infiltration, even as far as the central lumen of the pineal follicles. the proliferation of these cells in the non-palt pineal compartments is greater than in the palt based on tritiated-thymidine incorporation studies and counts of mitotic figures. the enhanced proliferation of lymphocytes in the pineal tissue suggests that the pineal cells produce blastogenic products that may also affect the differentiation of the lymphocytes. these results are additional evidence that lymphocytes traffic regularly between palt and the pineal follicles. the bu-1+ cells observed within the pineal follicles are most likely microglia/macrophages because their distribution and ramified morphology resemble the microglia/macrophages described in the mammalian pineal gland. to our knowledge, this is the first description of these cells in the avian pineal gland and, as such, represent ideal candidates as cellular interfaces between the immune system and the neuroendocrine pinealocytes. in contrast to most other parts of the brain, microglia/macrophages in the pineal gland are constitutively active as evidenced by the expression of mhc class ii. because these cells are actively involved in antigen presentation, and secrete potent cytokines, they have been implicated in the regulation of pinealocyte differentiation and neuroendocrine functions. tsai and mcnulty demonstrated in the rat that pineal microglia regulate pinealocyte neurite length and the production of serotonin in vitro. the production of melatonin could also be stimulated in vivo by treatment with il-1β, presumably produced by these activated microglia/macrophages. more recently, jiang-shieh et al. demonstrated that pineal microglia/macrophages are in constant surveillance of blood-borne pathogens and presumably other serum-derived substances. day-night differences in the expression of the bu- epitope in microglia/macrophages are consistent with their hypothesized role as interfaces in the interaction with palt lymphocytes. increased expression of bu- occurred at the dark:light interphase, the time immediately following when cells were exposed to the highest levels of melatonin, which is produced during the dark phase. the time-point corresponded to the rise in area density of tcrαβ/vβ + cells in the palt. this temporal relationship may be explained by increased secretion of microglial cytokines and chemokines assuming that increased expression of bu- represents activation of the microglia. the terminology used to describe microglia in the pineal gland is complicated by the fact that this organ is outside the blood-brain barrier. although perry and gordon described similar populations of cells in the choroid plexus and meninges as macrophages, we include the term "microglia" because the pineal gland is part of the brain, a terminology adapted by other authors. however, it is not clear whether these cells are undifferentiated microglia or resident macrophages derived from circulating monocytes. in addition to b-cells, the bu- antibody stains cells of the monocyte line and recognizes a highly glycosylated transmembrane protein that has no similarity to known mammalian markers. the bu- antibody has also been shown to stain microglia within the sensory epithelium of the ear. the functional significance of lymphocyte trafficking in the pineal gland has yet to be determined, although there are two overall working hypotheses, which are not mutually exclusive. one hypothesis addresses the mechanisms involved in the recruitment of lymphocytes to the gland and their possible differentiation to form distinct subpopulation, which are involved in immunological surveillance of the cns including the cerebrospinal fluid. in many species, the gland and the palt are juxtaposed to the choroid plexus, which produces csf. lymphocytes have been shown to migrate through the choroid plexus and enter the csf at these locations. the second hypothesis addresses immunological mechanisms regulating the neuroendocrine functions of the gland. as noted above, several studies have shown that immune-derived cytokines modulate both neural and neuroendocrine activities. it is therefore conceivable that lymphocyte trafficking in the pineal regulates the circadian production of melatonin. this potential circuit could represent a direct feedback of the immune system on a gland that is known to regulate immune functions.  in conclusion, this study has provided a detailed quantitative description of pineal-associated lymphocyte phenotypes at two time points over the 24-hour light:dark cycle. our results show that lymphocytes comprise a significant component of pineal tissue in this species. the data further suggest that cd8+/ tcrγδ+ cells are preferentially recruited to the pinealocytes and that pineal-lymphocyte interactions are dynamically linked to the light:dark cycle. pineal microglia/macrophages are proposed to play an important role in these interactions through their secretion of cytokines and chemokines. the unique accumulation of lymphocytes in this part of the brain has important functional implications with regard to homeostatic mechanisms of neuro-immune interactions, especially those involving circadian components of both the immune and nervous systems and the possible role of these cells in immune surveillance in other parts of the cns. further studies of the cellular and molecular mechanisms of these homeostataic pineal-lymphocyte interactions will provide a better understanding of disease processes in the nervous system. 
the pineal gland of the chicken contains large accumulations of lymphocytes that suggest an important role related to homeostatic circadian neuro-immune interactions. the purpose of this initial study was to characterize the lymphocyte subsets in the pineal gland and quantitate the distribution and frequency of lymphocyte phenotypes at two time points over the 24-hour light:dark cycle. our results further suggest that these interactions have a temporal component related to the 24-hour light:dark cycle and that cd8+ and tcrγδ+ t-cells are preferentially recruited to the pineal follicles. effects of the 24-h light:dark cycle on these lymphocyte-pineal interactions were suggested by an increase in the area of palt, a decline in the density of tcrαβ/vβ1+ cells, and a decline in the area density of bu-1+ microglia at the light:dark interphase compared to the dark:light interphase. the degree of lymphocyte infiltration in the pineal suggests novel mechanisms of neuro-immune interactions in this part of the brain. pineal microglia/macrophages were common and represent an important candidate for mediating these lymphocyte-pineal interactions via secretion of cytokines and chemokines. however, the ratios of cd8+/cd4+ and tcrγδ+/tcrαβ/vβ1+ within the pineal tissue were each 1: in contrast to the palt where the ratios of cd8+/cd4+ and tcrγδ+/tcrαβ/vβ1+ each approximated 1: bu-1+ cells were only rarely seen in the pineal interstitial spaces, but ramified bu-1+ microglia/macrophages were common in the pineal follicles. palt comprised approximately 10% of the total pineal area. image analysis of immunocytochemically stained sections showed that the majority of lymphocytes were cd3+ with the remaining 20% comprising b-cells and monocytes, which tended to distribute along the periphery of the palt. all of the t-cell phenotypes were commonly found within the interfollicular septa and follicles of the pineal gland.
 homeostatic trafficking of lymphocytes in the brain has important relevance to the understanding of cns disease processes. the pineal gland of the chicken contains large accumulations of lymphocytes that suggest an important role related to homeostatic circadian neuro-immune interactions. the purpose of this initial study was to characterize the lymphocyte subsets in the pineal gland and quantitate the distribution and frequency of lymphocyte phenotypes at two time points over the 24-hour light:dark cycle.  palt comprised approximately 10% of the total pineal area. image analysis of immunocytochemically stained sections showed that the majority of lymphocytes were cd3+ with the remaining 20% comprising b-cells and monocytes, which tended to distribute along the periphery of the palt. t-cell subsets in palt included cd4+, cd8+, tcrαβ/vβ1+, and tcrγδ+. all of the t-cell phenotypes were commonly found within the interfollicular septa and follicles of the pineal gland. however, the ratios of cd8+/cd4+ and tcrγδ+/tcrαβ/vβ1+ within the pineal tissue were each 1: in contrast to the palt where the ratios of cd8+/cd4+ and tcrγδ+/tcrαβ/vβ1+ each approximated 1: bu-1+ cells were only rarely seen in the pineal interstitial spaces, but ramified bu-1+ microglia/macrophages were common in the pineal follicles. effects of the 24-h light:dark cycle on these lymphocyte-pineal interactions were suggested by an increase in the area of palt, a decline in the density of tcrαβ/vβ1+ cells, and a decline in the area density of bu-1+ microglia at the light:dark interphase compared to the dark:light interphase.  the degree of lymphocyte infiltration in the pineal suggests novel mechanisms of neuro-immune interactions in this part of the brain. our results further suggest that these interactions have a temporal component related to the 24-hour light:dark cycle and that cd8+ and tcrγδ+ t-cells are preferentially recruited to the pineal follicles. pineal microglia/macrophages were common and represent an important candidate for mediating these lymphocyte-pineal interactions via secretion of cytokines and chemokines. 