Hormonal control of neutrophil activity in the rat vagina

chemotactic

THOMAS W. JUNGI, HUGO 0. BESEDOVSKY, ERNST SORKIN, AND MARTIN SCHARDT Schweizerisches Forschungsinstitut, Medizinische Abteilung, 7270 Davos, Switzerland; Facultad de Ciencias Medicas, Instituto de Fisiologia, Rosario, Santa Fe 3100, Argentine; and James A. Baker Institute for Animal Health, Cornell University, Ithaca, New York 12853

JUNGI, THOMAS W., HUGO 0. B~SEDOVSKY ,ERNST~ORKIN, AND MARTIN SCHARDT. Hormonal control of neutrophil chemotactic activity in the rat vagina. Am. J. Physiol. 233(l): R59-R65, 1977 or Am. J. Physiol.: Regulatory Integrative Comp. Physiol. 2(l): R59-R65, 1977. -A sexual-hormonedependent neutrophil chemotactic factor(s), operative under physiological conditions, is described. Rat vaginal washouts were shown to be both chemotactic and chemokinetic for rat neutrophils in vitro, whereas macrophages were not attracted. Peak activity was observed at the end of estrus and preceded maximal neutrophil infiltration in the vagina. In order to mimic these events, gonadectomized animals were treated with estradiol for a week. They showed a similar peak of chemotactic activity 30-36 h after estradiol withdrawal, accompanied by a massive neutrophil accumulation. These data suggest that a decrease in estradiol level permits the expression of the chemotactic signal. There was no evidence for chemotaxis and/or migration inhibitors before and during estrus or during long-term estradiol treatment of gonadectomized rats. Induced neutrophil accumulation in the peritoneal cavity and chemotactic responsiveness of these cells in vitro were similar in all stages during the estrus cycle. Estradiol, progesterone, testosterone, and hydrocortisone neither promoted nor significantly inhibited the neutrophil migratory behavior over a wide range of concentrations. Our experiments suggest that the periodic neutrophil accumulation in the rat vagina after estrus is triggered by locally expressed chemotactic mechanisms that are controlled by sexual hormones. The data provide the first evidence that hormonal changes can control chemotactic factors and thus indirectly control cell migration. cell migration;

estradiol;

CELL MIGRATION

plays

estrus

cycle

an important

role in embryogene-

sis, regeneration, inflammation, and other processes. Hormones can potentially control many processes related to migratory behavior of cells. However, to the best of our knowledge, regulation of cell migration by hormones under physiological conditions has not been studied. In rodents, neutrophil leukocytes appear periodically in the vagina after the estrus stage and disappear at the end of diestrus (7). This phenomenon is so significant in rats and mice that it has been used as an indicator of pregnancy (Allen-Doisy test (1)). The possibility was considered by us that this sexual-hormonedependent neutrophil migratory process involves chemotaxis, i.e., directional movement of cells toward chemi-

cal stimuli (11, 12). Evidence will be presented for periodically appearing neutrophil-specific chemotactic activity in vaginal secretions during the estrus cycle, suggesting a hormonal control of chemotactic mechanisms. MATERIALS

Animals.

AND

METHODS

Specific pathogen-free Wistar albino rats from Tierfarm Fullinsdorf, and maintained under conventional condi-

(200-300 g) were obtained

Switzerland tions. Vaginal secretions. Vaginas were washed with exactly 0.5 ml of Gey’s balanced salt solution (GBSS). In order to eliminate possible circadian variance, all washouts were collected between 9 A.M. and 11 A.M. except when specified otherwise. Usually, for each collection, samples from 40 to 90 rats were processed. Stages of the sexual cycle were determined according to Long and Evans (7) by microscopic inspection without staining. The histological composition was recorded, using an arbitrary score from 0 to + + + +. Washouts were centrifuged for 10 min at 300 x g, and the supernatant was immediately frozen and kept at -7OOC until use for chemotaxis experiments. (Storage at -20°C resulted in progressive loss of chemotactic activity). Seven or more fresh or fresh frozen washouts of identical stages were pooled and Millipore filtered (O.&&pm pore size) prior to further use in cell migration experiments. Similar washouts were obtained from gonadectomized animals before, during, and after a 1-wk treatment with estradiol. Washouts collected at identical intervals after gonadectomy, or after onset of estradiol regime, which showed identical histological patterns, were pooled for migration assays. Indicator cells for chemotaxis, chemokinesis, and migration modulation assays. The effect of vaginal secretions upon phagocyte migration was measured by use of indicator cells derived from induced peritoneal exudates. Indicator rat neutrophils were induced by intraperitoneal (ip) injection of 10 ml sodium caseinate (3.5% wt/vol in saline; Bender & Hobein, Zurich, Switzerland) lo-12 h prior to harvest. After having established that chemotactic responsiveness of peritoneal neutrophils did not vary during estrus cycle (see Table 3), exudates of females were also included. Exudates consisted of 75 90% neutrophils. These were the only cells which could

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R60 penetrate 3-pm filters within the incubation time. Exudates were collected with GBSS. The pooled cells were spun at 200 x g, washed twice with GBSS, and resuspended in GBSS containing 2% wt/vol of human serum albumin (HSA; Swiss Red Cross Blood Transfusion Center, Berne, Switzerland) at a concentration of 4 x lo6 neutrophils/ml. Presence of 0.52% HSA was indispensable for reproducibility of results. For chemotaxis and migration modulation experiments, aliquots of 5 x lo6 neutrophils were dispensed into siliconized glass tubes and spun, and the pellet was resuspended in the desired material. Indicator macrophages were collected 3 days afier ip injection into male rats of 10 ml peptone (10% wt/vol in saline; Pepton fur Bakteriologie, Fluka, Buchs, Switzerland). They were processed as outlined for neutrophils and suspended at a concentration of 3 x lo6 macrophages/ml in HSA-containing medium. Peptone induced exudates consisted of 80-90% macrophages, the rest being mainly neutrophils. Measurement of chemotaxis. Chemotactic activity of pools of vaginal washouts was measured with a modified Boyden chamber (3, 6), which is composed of two compartments separated by a Millipore-type membrane (Sartorius Membranfilter, Gottingen, West Germany), with a pore size big enough to allow leukocytes to penetrate the filter by amoeboid movement (3 pm for neutrophils, 8 pm for macrophages). The test agent, 0.5 ml, was applied to the lower compartment with a syringe, and 0.5 ml of a suspension of indicator phagocytes was added to the upper-compartment. Before filling, the pH of both the test solution and the cell suspension was adjusted to 7.3. The capped chambers were incubated (4 h for neutrophils, 5 h for macrophages) at 37OC. Chambers were reversed midway during the incubation in order to avoid detachment of migrated cells from the filter surface (6). After incubation, chambers were dismantled, filters were fixed in ethanol (96%), and cells were stained with Weigert’s hematoxylin, dehydrated, cleared, and mounted on slides, as described elsewhere (6). Cells that h a d completely penetrated the filter and accumulated at its lower side within the incubation period were counted in four microscopic fields (magnification x240) and averaged. Duplicate or triplicate chambers were run. Known strong attractants, such as immune complex, activated-then-heated rat serum (5%) or casein Hammarsten (1% or 0.5%) for neutrophils, and 100% heated (30 min at 56OC) rat serum for macrophages, served as positive controls, as described elsewhere ((6) and Jungi and McGregor, manuscript submitted for publication). Chambers containing GBSS only in the lower compartment were included as negative controls. Chemotaxis of rat neutrophils and macrophages toward estradiol, progesterone, testosterone, and hydrocortisone at various concentrations was assayed similarly. Hormone dilutions were prepared with GBSS from stock solutions made up in ethanol. Control chambers contained a strong attractant in the lower compartment to which the same final concentration of ethanol was added as present in hormone dilutions. Measurement of chemokinesis. The same test system

JUNGI,

BESEDOVSKY,

SORKIN,

AND

SCHARDT

as described for chemotaxis measurement was used for chemokinesis. But for comparison between directional movement (chemotactic response) and increase of random motility (chemokinetic response), various concentrations of vaginal material were applied to the lower compartment and combined with the same or another concentration of the same washout pool in the upper (cell-containing) compartment. Thus, migration through filters containing the chemotactic material as a positive gradient (concentration beneath filter greater than that above filter) or a negative gradient (concentration beneath filter less than that above filter) could be compared with migration in the absence of a chemotactic gradient. Modulation of cell migration in vitro by diluted vaginal washouts or hormones. To test for presence of factors inhibiting neutrophil migration and/or chemotactic stages were response, vaginal washouts of different mixed with various amounts of medium. To 5 x log neutrophils suspended in 1.9 ml of these dilutions, 0.1 ml of 20% HSA was added. To another 1.9 ml of the vaginal dilutions, 0.1 ml of activated serum was added. The latter mixtures were dispensed into the lower compartments of triplicate chemotaxis chambers. Corresponding mixtures containing the indicator cells were added to the upper compartment. Chemotactic migration in the presence of various dilutions of vaginal washouts could thus be compared with the cellular response in the absence of vaginal constituents. It was expressed in percent of the unmodulated response by use of the formula actual chemotactic

response,

T-N % = ~p - N x loo

where T = cells per field on test filter, N = cells per field on negative control filters, and P = cells per field on positive control filters. Similarly, a direct effect of female sexual hormones upon chemotactic leukocyte migration toward activated serum (5%) and casein (1%) was assayed by including estradiol, progesterone, and estradiol-progesterone mixtures at various concentrations in both compartments. A chemotactic response was expressed as percent of migration in absence of hormones by use of the above formula. Neutrophil accumulation at an infZammatory site. Four hours after ip injection of 10 ml sodium caseinate (3.5% in saline), individual peritoneal exudates from large groups of female rats (20-24 per day) were collected, and neutrophil yields were related to the stages of estrus cycle. The latter were determined as outlined above at time of exudate collection. Neutrophil chemotactic responsiveness in vitro. The above-described cells derived from inflammatory exudates were tested for their chemotactic responsiveness toward strong attractants (5% activated serum or 0.5% casein). N.umbers of neutrophils per field were related to the stages of sexual cycle of the cell donors. This allowed comparison of intr insic chemotactic responsi veness of peritoneal exudate neutrophils obtained from donors at

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HORMONAL

CONTROL

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other cells present); metaestrus early (with many cornified cells, but typically also round epithelial cells present, appearance of neutrophils); metaestrus late (very high number of neutrophils, variable amount of round cells); and diestrus early (with neutrophils, following late metaestrus). Diestrus with low cell numbers were pooled separately; washouts from animals with disturbed cycles were discarded. It can be seen in Fig. 1 that the relatively low attractive activity of vaginal washouts increases considerably at the end of estrus, peaks in late estrus or early metaestrus, then decreases quickly, reaching a minimum at late metaestrus. Thus, peak activity precedes maximal neutrophil accumulation in the vagina, suggesting that the latter is related to the neutrophil attractive force of vaginal secretions. For these experiments, it was tacitly assumed that the vaginal activity involved chemotaxis (activity increasing directionality of movement) rather than chemokinesis (activity influencing velocity of movement). However, it was necessary to devise experiments that would distinguish between the two types of chemoattractive mechanisms. Combinations of various concentrations of estrus washouts were used in the two compartments of chemotaxis chambers, as shown in Table 1. If the substance were chemokinetic only, estrus washouts, being present as a negative gradient or in equal concentrations in both compartments, would also cause neutrophil accumulation on the lower filter side. It is shown in Table 1 that the strongest migration occurs when the washouts are present as a positive gradient. A negative gradient of active washouts does not lead to chemoattraction in vitro, whereas equal concentrations of the same active material (no gradient) caused intermediate migration. This indicates that the chemoattractant present in the vagina during late estrus and metaestrus is chemotactic, but in addition also chemokinetic. In the following, the term chemotaxis is used

different stages of the sexual cycle. Gonadectomy . In rats that were anesthetized with ether, ovaries were removed by standard surgical procedures. Wounds were closed with interrupted sutures, and animals were kept isolated 3-5 days. Estradiol regime. Gonadectomized rats received 3 pg of 17.beta estradiol im twice daily for 7 days. In some experiments, one daily dose of 5 rug was given instead. The hormone was obtained from Fluka, Buchs, Switzerland, and a stock solution made up in ethanol was diluted in saline to the desired concentration. RESULTS

Sexual-cycle-stage-dependent activity of vaginal washouts.

neutrophil

chemotactic

It was assumed that periodic neutrophil accumulation in the rodent vagina is a local hormone-mediated phenomenon. Vaginal washouts obtained during the different stages of the sexual cycle were tested for their neutrophil attractive properties to ascertain that chemotaxis was involved. Pilot experiments showed that vaginal washouts contained dose-dependent, stage-specific chemotactic activity for neutrophils. Paradoxically, the estrus stage, which is free of neutrophils, contained most of the activity. In order to study a possible relationship of chemotactic activity in vitro and neutrophil accumulation in vivo, vaginas of large groups of rats (60-90) were washed out at 12-h intervals (9-11 A.M. and P.M.) over 5 days, and the cellular composition of washouts was recorded for each rat individually. This allowed accurate determination of substages of the sexual cycle. For chemotaxis assay, individual washouts were pooled as follows: diestrus late (preceding proestrus); proestrus early (containing very few cells of either type); proestrus late (containing merely round epithelial cells); estrus early (containing round epithelial cells and cornified cells); estrus late (very rich in cornified cells, no

I....-\ .. I--o-

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-

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0

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FIG. 1. Stage-specific neutrophil chemotactic activity of rat vaginal washouts during estrus cycle. Chemoattraction by pools of 7-8 corresponding washouts and various dilutions thereof at different stages of the sexual cycle is shown. Peak activity is found at end of estrus and precedes neutrophil accumulation. By use of an arbitrary score, from 0 to + + + + , relative neutrophil numbers are visualized by plotting means of score numbers of the individual washouts pooled for chemotaxis test of each group. (-) washout pool, undiluted (0.5 ml per rat vagina); (----) washout pool, diluted pool, diluted 1:4. 1:2; ( -----) washout

EC.-

diestrus early

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R62

JUNGI,

1. Chemotactic and chemokinetic activity Late estrus -early metaestrus vaginal washouts for peritoneal neutrophils in vitro TABLE

of pooled

Upper Compartment* Lower Compartment?

Vaginal Vaginal 1:3 GBSS

washout

Vaginal

262.0

washout,

washout

+ 35.0 nd

27.3

IT 8.5

Vaginal washout, 1:3

nd 248.7

+ 49.6

34.6

k 11.5

GBSS

633.3

k 59.3

416.0

+ 66.4

9.3 2 3.0

Table entries are number of cells per field (mean * SD); nd = not done. * 0.1 vol/vol of 20% HSA added after cells have been resuspended in test material. TO.1 vol/vol of GBSS added to test material (pool of 0.5-ml vaginal washouts from rats in late estrus or early metaestrus).

BESEDOVSKY,

SORKIN,

AND

SCHARDT

for the activity observed in vitro. In addition, it could be argued that hormonal concentrations in the circulating blood are much lower than at the site of periodic neutrophi1 accumulation. Therefore, sexual hormones were also tested for their direct chemoattractive properties. Estradiol or progesterone at concentrations ranging from 10 to 0.001 pg were applied to the lower compartment of chemotactic chambers. For comparison, testosterone and hydrocortisone were included at the same concentrations. All hormones tested were devoid of chemotactic activity for neutrophils and macrophages over the whole concentration range. Also in migration modulation tests, chemotaxis of polymorphonuclear leukocytes was not influenced by several hormones. When progesterone (1 ,ug-1 mg/ml), estradiol (28 ng-28 pg/ ml), or hydrocortisone (15 pg-15 rig/ml) was included in both compartments, the migratory behavior of neutrophils toward activated serum (5%) was not influenced. This suggested that cyclical neutrophil accumulation in female rats is a local phmomenon that is indirectly mediated by hormones. Chemotactic activity of vaginal washouts from rats treated with estradiol after gonadectomy. The foregoing experiments suggest that neutrophil chemoattractive activity triggers neutrophil accumulation in the vagina after estrus. On the other hand, the measured chemotactic activity could be due to the presence of cornified cells. To explore these possibilities, washouts were examined microscopically in rats after gonadectomy. Washout of high or low neutrophil contents were pooled for chemotaxis tests. It was found that vaginal chemo-

interchangeably with chemoattraction for describing both chemotactic and chemokinetic activity of vaginal fluid. Chemotactic activity of vaginal washouts is ceLL specific. Tests utilizing rat peritoneal macrophages revealed cell specificity of these chemoattractants in the vaginal washouts. Only O-l macrophage per field was attracted in vitro, although these cells had responded vigorously to undiluted heated rat serum as attractant (128 macrophages per field). This finding correlates well with the presence of neutrophils but not macrophages in the vaginal lumen. Do changes in blood Levels of sexual hormones affect the acute inflammatory response? The preceding experiments made it evident that the sexual cycle affects TABLE 2. Neutrophil inflammatory response chemoattractive activity locally, but it was also of interfollowing intraperitoneal stimulation est to ascertain the systemic consequences of hormone changes. An in’vestigation was made to determine Stage of Sexual Cyclet Peritoneal Neutrophils per Exudate* x lo8 whether neutrophils accumulate at different rates in Proestrus 88.4 _+ 60.1 (15) acute inflammatory exudates, depending on the sexual Estrus 83.0 + 40.5 (28) Metaestrus 82.9 t 56.4 (13) stage of the animal, that is, when the various sexual 88.2 + 39.2 (34) hormones are present in different concentrations in Diestrus blood. Table 2 shows that neutrophil leukocyte yields Mean of all exudates 85.8 + 45.5 (90) obtained from peritoneal exudates 4 h after caseinate Values are means * SD, with number of exudates tested in pareninjection vary considerably from animal to animal, but theses. * Cells were harvested 4 h after ip injection of 10 ml of there was no significant difference in the inflammatory 3.5% sodium caseinate. t Sexual cycle stage was determined at time of exudate collection. response between the stages of the sexual cycle of the donor at the time of exudate collection. Thus, the different levels of sexual hormones in the blood are not re- TABLE 3. Chemotactic responsiveness of lated to the neutrophil accumulation at an acute inflamperitoneal exudate neutrophils matory site. Chemotactic Response* Do changes in blood level of sexual hormones affect Stage of Sexual Cyclet p Attractant: activated rat sethe intrinsic chemotactic responsiveness of peritoneal Attractant: casein, 0.5% rum, 5% from exudate neutrophils ? The above cell populations Proestrus 113.0 t 83& (6) 95.2 + 97.0 (6) individual females were also tested in vitro for their Estrus 125.4 t 56:7 (12) 91.6 + 33.3 (12) chemotactic responsiveness toward two different stimMetaestrus 112.4 t 35.2 (5) 97.8 + 45.1 (5) uli, namely 0.5% caseinate and 5% activated heated Diestrus 140.4 -+ 56.0 (19) 90.6 + 45.0 (19) serum (Table 3). No correlation between chemotactic Mean of all cell 128.9 t 58.0 (42) 92.4 + 51.9 (42) responsiveness in vitro and the cell donor stage of the populations sexual cycle was noted. Values are.number of cells per field (mean f SD), with number of Influence of sexual hormones on migration in vitro. exudates tested in parentheses. * Exudates harvested 4 h after ip Since the chemotactic activity of vaginal washouts injection of 10 ml of 3.5% sodium caseinate were tested individually proved to be estrus-cycle-dependent, it seemed possible for their chemotactic response toward standard stimuli. t Sexual that one of the sexual hormones was directly responsible cycle stage was determined at time of exudate collection. Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (130.237.122.245) on August 18, 2018. Copyright © 1977 American Physiological Society. All rights reserved.

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tactic activity was similar in all washouts of gonadectomized rats irrespective of time of collection or of neutrophil content. Ten to fourteen days after gonadectomy, animals were given daily 5-6 pg estradiol im, resulting in disappearance of neutrophils and appearance of cornified cells, as in estrus. Washouts were taken at various intervals and inspected. Those collected at identical times after the first estradiol injection and having similar cytological appearance were pooled, and the supernatants were assayed for chemotaxis. The results obtained (not shown here) indicated that, unlike normal estrus washouts, those obtained 70-114 h a&er the first estradiol injection contained little chemotactic activity, although the histological pattern is not distinguishable from estrus. It is concluded that the chemotactic activity of normal washouts is not a simple consequence of the appearance of corniced cells. During prolonged estradiol treatment of gonadectomized rats, two phases of increased chemotactic activity can reproducibly be registered: one shortly after onset of estradiol treatment, which is not followed by neutrophil accumulation in vivo (data not shown); and the other after withdrawal of estradiol, which is accompanied by massive neutrophil appearance and subsequent disappearance of cornified cells, usually 36 h after the last estradiol injection. The kinetics of chemoattractive activity and neutrophil accumulation after estradiol withdrawal, documented in Fig. 2, resemble the phenomena at the end of estrus in normal animals (Fig. 1). This appearance of chemotactic activity in normal washouts is correlated with the subsequent neutrophil accumulation in vivo and is triggered by the decreased estradiol level. On the other hand, these results provide evidence that regulatory mechanisms _other than chemoattraction prevent neutrophils from accumulating in the vagina when cornified cells are present, since despite de-

monstrable chemotactic activity, no neutrophils entered the vagina shortly after onset of estradiol treatment. It was considered therefore that inhibitors might affect chemotaxis and/or random migratory behavior of neutrophils. Chemotactic migration of peritoneal exudate neutrophils is not inhibited by vaginal washouts. Different pools of vaginal washouts were tested for their inhibitory effect on cellular response in a chemotaxis test. For comparing chemotactic migration toward an unrelated stimulus in the presence and absence of vaginal fluid, migration modulation experiments (see MATERIALS AND METHODS) were performed. Vaginal washout pools at neither stage of the sexual cycle inhibited chemotactic migration in vitro (Fig. 3; other data not shown). Also washouts from rats with and without estradiol after gonadectomy (Fig. 3) were inactive in this respect. These data do not support the view that a migration and/or chemotaxis inhibitor in the vagina is responsible for absence of neutrophils during proestrus and estrus and during long-term estradiol treatment of gonadectomized rats. DISCUSSION

The periodic accumulation of neutrophil leukocytes in the rodent vagina is governed by a subtle interplay of the female sexual hormones, the estrogens and progesterone. On the other hand, the ability of neutrophils to migrate directionally toward chemical stimuli in vitro may be related to neutrophil accumulation in inflammatory foci. Therefore, we sought to determine whether chemotactic mechanisms are involved in the periodic appearance and disappearance of neutrophils in the vagina during the estrus cycle of the rat and the way in which this process is linked to the hosts’ hormonal sta-

-200

FIG. 2. Appearance of chemotactic activity in vaginal washouts after termination of 5-day estradiol treatment of gonadectomized rats. Peak activity parallels neutrophil influx 36 h after estradiol withdrawal. Details are as in Fig. 1.

-umdid Mad

I2h

24h

time

after

Ill-dJ 72h

estrad

i ol

injection

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R64

JIJNGI,

wo,h out

pool Of

early rrtrw

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rsttodiol

pool

trootod

rots

FIG. 3. Absence of neutrophils in vagina during proestrus and estrus and during estradiol treatment of gonadectomized rats cannot be ascribed to an inhibitor of neutrophil migratory response. Cells suspended in vaginal washouts obtained from these stages were not inhibited in their migration towards 5% activated serum compared to cells suspended in GBSS only (100% migration).

tus. It was found that rat vaginal washouts contain dose-dependent, stage-specific chemotactic activity for neutrophils, but washings of neither stage attract macrophages in vitro. The activity proved to be both chemotactic (increasing directionality) and chemokinetic (increasing motility). The attractant was highly active compared with known other agents, in spite of the considerable dilution used to procure the material from the vagina. Kinetic studies indicated that peak chemotactic activity was found in late estrus-early metaestrus, preceding maximal neutrophil accumulation by lo-12 h (Fig. 1). This suggests that neutrophil localization in the vagina is triggered by a chemoattractant which appears at the end of estrus. This notion was confirmed in experiments with gonadectomized rats that were examined before, during, and after estradiol treatment. Chemotactic activity of washouts from gonadectomized rats, containing variable amounts of neutrophils, was as low as in diestrus. Washouts from gonadectomized rats treated with estradiol for a week showed two peaks of activity. Between these two peaks, chemotactic activity was moderate despite the presence of cornif’ied cells throughout. This suggested that presence of these cells is not causally linked to the chemotactic activity of vaginal washouts. Furthermore, the described phenomena occurring at the end of estrus can be mimicked by estradiol withdrawal in gonadectomized rats. This is compatible with the findings of Shaikh (10) that during the estrus cycle of the rat estradiol concentration in the blood peaks in proestrus and decreases during estrus. Appearance of chemotactic activity in the vagina seems to be a locally occurring hormone-triggered phenomenon. Neither extent of neutrophil accumulation in an acute inflammatory exudate nor chemotactic responsiveness of neutrophils in vitro proved to be correlated

BESEDOVSKY,

SORKIN,

AND

SCHARDT

to the stage of the sexual cycle. Accordingly, it seems safe to conclude that migratory capability of peripheral blood neutrophils and peritoneal exudate neutrophils is not directly affected by the hormones governing the sexual cycle. In contrast, other parameters such as mast cell count and histamine content have been reported to vary considerably during the estrus cycle (5). Moreover, our results contrast with those on human peripheral blood neutrophils which, when taken at different days of the sexual cycle and tested in vitro for their chemotactic response, showed a decrease during menstruation time (Frei, personal communication). Preliminary experiments suggest that the reported vaginal chemotactic activity demonstrated in vitro promotes neutrophil accumulation in vivo. Intraperitoneal injection of estrus and metaestrus washouts resulted in accumulation of considerably more neutrophils than diestrus washouts or medium only. Intravaginal application of active washouts to gonadectomized rats also induces increased neutrophil localization. Interestingly, active washouts do not attract neutrophils in the vaginas of gonadectomized rats treated with estradiol. Accordingly, in spite of a chemotactic signal at the beginning of the hormone treatment, neutrophils did not migrate into the vagina. This could be due to anatomical characteristics of the vaginal wall in that stage (Sellers, personal communication). However this may be, search for an inhibitor in proestrus and estrus washouts decreasing a cellular response in vitro has hitherto been unsuccessful. Similarly, during late proestrus and diestrus and during estradiol treatment of gonadectomized rats, no inhibition of chemotactic migration by vaginal secretion could be demonstrated in vitro. The mechanisms which prevent neutrophils from accumulating in the vagina when estradiol levels are increasing are thus still unclear. The origin and nature of the vaginal chemotactic factor(s) for neutrophils remain to be determined. It is suggested that chemotactic activity is produced by the epithelial cells of the vagina under the specific triggering influence of sexual hormones during the estrus cycle. Although presently under study, it is considered unlikely that products of the vaginal flora are responsible for the observed chemotactic activity. It is noteworthy in this context that germ-free mice have a normal vaginal neutrophil response induced by progesterone (2). Preliminary experiments as well as cell specificity seem to argue against involvement of complement split products although this has to be further substantiated. Moreover, the hormonally controlled intrinsic factor(s) for neutrophil accumulation in vivo differs in many ways from that described by Maroni et al. (8, 9) and Clark and Klebanoff (4). These authors demonstrated in vitro neutrophil chemotactic activity of spermatozoa and seminal fluid in the presence of serum and plasma. In conclusion, we have shown here for the first time, that hormonal changes can trigger locally the release of cell-specific chemotactic factor(s) in the vagina. It is suggested that vaginal chemotactic activity is related to neutrophil migration into the vaginal lumen after estrus. It will be of interest to look for other examples that hormones can control the migration of cells.

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The excellent technical assistance by Mr. Bosco Koncar and Mrs. Ruth Jungi is gratefully acknowledged. We also thank Miss Helene Kreuzer for preparing the manuscript and figures. This work was supported by Schweizerischer Nationalfonds zur

Fiirderung 3.600.75

and

Received

for

der wissenschaftlichen by F. Hoffmann-La publication

Forschung, Roche, Basel.

16 September

Grants

36750.72

and

1976.

REFERENCES 1. ALLEN, E., AND E. A. DOISY. An ovarian hormone. Preliminary report on its localization, extraction and partial purification, and action in test animals. J. Am. Med. Assoc. 81: 819-821,1923. 2. BEAVER, D. L. The hormonal induction of a vaginal leukocytic exudate in the germ-free mouse. Am. J. Pathol. 37: 769-773, 1960. 3. BOYDEN, S. V. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exptl. Med. 115: 453-466, 1962. 4. CLARK, R. A., AND S. J. KLEBANOFF. Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors. J. Immunol. 117: 1378-1386, 1976. 5. JAQUES, R., AND M. RUECG. Age and sex differences in mast cell count and histamine content. Agents and Actions. 1: 144-167, 1970. 6. JUNGI, T. W. Assay of chemotaxis by a reversible Boyden cham-

7. 8.

9.

10.

11. 12.

ber eliminating cell detachment. Intern. Arch. Allergy Appl. Immunol. 48: 341-352, 1975. LONG, J. A., AND H. M. EVANS. The oestrus cycle in the rat and its associated phenomena. Mem. Univ. Calif. 6, 1922. MARO.NI, E. S., D. N. K. SYMON, AND P. C. WILKINSON. Chemotaxis of neutrophil leucocytes towards spermatozoa and seminal fluid. J. Reprod. Fertility 28: 359-368, 1972. MARONI, E. S., AND P. C. WILKINSON. Selective chemotaxis of macrophages towards human and guinea-pig spermatozoa. J. Reprod. Fertility 27: 149-152, 1971. SHAIEH, A. A. Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. BioZ. Reprod. 5: 297-307, 1971. SORKIN, E. (Editor). Chemotuxis: its Biology and Biochemistry. Basel: Karger, 1974. WILKINSON, P. C. Chemotuxis and InfZammution. Edinburgh: Churchill-Livingstone, 1974.

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Hormonal control of neutrophil chemotactic activity in the rat vagina.

Hormonal control of neutrophil activity in the rat vagina chemotactic THOMAS W. JUNGI, HUGO 0. BESEDOVSKY, ERNST SORKIN, AND MARTIN SCHARDT Schweize...
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