Immunoendocrine mechanisms in mammary tumor progression: Direct prolactin modulation of peripheral and preneoplastic hyperplastic-alveolar-nodule-infiltrating lymphocytes

We have previously shown that the immunoregulatory function of prolactin may play a role in the progression of the mouse mammary preneoplastic hyperplastic alveolar nodule (HAN) line C4 to carcinoma. In this study we investigated the direct effect of prolactin on lymphocytes isolated from normal and C4-HAN-bearing mice. In addition, we tested the effect of ovariectomy on prolactin/lymphocyte interaction to see whether, as has been reported in rats [Mukherjee P., Hymer W. C. (1992) Prog Neuroendocrinol Immunol 5: 108; Viselli S. M. et al. (1991) Endocrinology 129: 983], removal of estrogen would enhance the response to prolactin in mice. Proliferation of splenocytes, lymph node cells and HAN-in-filtrating lymphocytes was stimulated by prolactin in a dose-responsive fashion. Ovariectomy did not alter this effect consistently. Cell-cycle analysis based on simultaneous staining of DNA and RNA revealed that prolactinstimulated lymphocytes progress through all phases of the cell cycle whereas anti-prolactin antiserum inhibits this stimulation. Two-color flow-cytometric analysis revealed the time-dependent induction of interleukin-2 (IL-2) receptor expression on both CD4⁺ and CD8⁺ cells by prolactin. Prolactin-treated lymphocytes also produced low yet detectable levels of bioactive IL-2 in a dose-and time-dependent fashion. Prolactin enhanced lymphocyte responsiveness to mitogens and showed a marked synergism at suboptimal concentrations. Pretreatment of splenocytes from HAN bearers with a high concentration of prolactin slightly enhanced natural killer (NK) activity; anti-prolactin antiserum reduced the NK lytic activity of poly(I)-poly(C)-activated splenocytes from HAN-bearing mice. Our results provide direct experimental evidence for the stimulatory effect of prolactin on lymphocyte function and IL-2-mediated lymphocyte proliferation and suggest a mechanism linking the endocrine system to immunomediated enhancement of HAN progression.     

Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons

Magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) show considerable plasticity during pregnancy and lactation. Prolactin receptors (PRL-R) have been identified in both these nuclei. The aim of this study was to investigate the cell type(s) expressing mRNA for the long form of prolactin receptor (PRL-R(L)) and to determine whether patterns of expression change during pregnancy and lactation. In addition, we examined effects of prolactin on excitability of oxytocin and vasopressin neurons. Sections from brains of nonpregnant, pregnant, and lactating rats were hybridized with an 35S-labeled probe to label PRL-R(L) mRNA together with digoxigenin-labeled probes to detect either oxytocin or vasopressin mRNA. In the SON, PRL-R(L) mRNA was predominantly colocalized with oxytocin mRNA, with over 80% of oxytocin neurons positive for PRL-R(L) mRNA. Very few (<10%) vasopressin neurons expressed PRL-R(L) mRNA. In the PVN, PRL-R(L) mRNA was also predominantly found in oxytocin neurons, and the proportion of PRL-R(L)-positive oxytocin neurons increased significantly during pregnancy and lactation. As in the SON, relatively few vasopressin cells contained PRL-R(L) mRNA. For in vivo electrophysiology, nonpregnant rats were anesthetized, and then extracellular single neuron activity was recorded in identified oxytocin and vasopressin neurons. After a period of baseline recording, the effect of prolactin (1 microg i.c.v.) on firing rate was examined. Prolactin treatment of nonpregnant rats induced a significant decrease in firing rates of oxytocin neurons. There was no effect of prolactin on the activity of vasopressin neurons. Together, these data provide strong evidence that prolactin directly and specifically regulates activity of oxytocin neurons.     

Upregulation of hepatic prolactin receptor gene expression by 17beta-estradiol following trauma-hemorrhage.

Although studies show protective effects of 17beta-estradiol (E2) or prolactin (PRL) treatment in male rats after trauma-hemorrhage (TH), the mechanism of the salutary effects of these agents remains unknown. Because E2 modulates PRL receptor (PRL-R) expression in the liver, we examined whether E2 treatment after T-H has any effects on hepatic PLR-R gene expression. Male Sprague-Dawley rats were subjected to trauma (i.e., 5-cm midline laparotomy) and hemorrhage (35-40 mmHg for 90 min) followed by fluid resuscitation (Ringer lactate) or sham operation and then treated with E2 (50 microg/kg body wt sc) or vehicle immediately before resuscitation. Liver samples were collected at 3 h thereafter, and PRL-R mRNA expression was determined by PCR. Liver expression of PRL-R short-form gene was unaffected by T-H, whereas that of the long-form gene was suppressed. Treatment of T-H rats with E2 significantly increased PRL-R short-form gene expression and normalized PRL-R long-form gene expression to sham levels. In the isolated hepatocytes, PRL-R short-form gene expression was predominant compared with the long-form gene. In contrast, only the short form was detected in Kupffer cells. In vitro treatment by E2 demonstrated an increase in the PRL-R long-form gene in hepatocytes, but E2 had no effect on PRL-R short-form gene expression in either the Kupffer cells or hepatocytes. Thus E2 treatment after T-H in males appears to directly upregulate PRL-R long-form gene expression in hepatocytes. However, the upregulation of the PRL-R short form might involve the interaction of multiple cell types in the liver.     

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.     

Human Lymphocyte Proliferation: Dna Synthesis Time

Abstract. The DNA synthesis time ( T _s) of lymphocytes from spleens and lymph nodes of patients with Hodgkin's disease was determined by the double labeling method. ³H-TdR was administered in vivo and removed tissues minced in ¹⁴C-TdR in vitro. Lymphocytes from patients with lymphosarcoma and reticulum cell sarcoma were studied in a similar manner. Lymphocytes were divided into A cells, small with non-basophilic cytoplasm, B cells, small with basophilic cytoplasm, C cells, large with non-basophilic cytoplasm, and D cells, large with basophilic cytoplasm.
The T _s of splenic lymphocytes, in four samples not containing Reed-Sternberg cells, in hours, was: B 11.3; C , 7.9; D , 8.4; combined, 8.8. the T _s of B lymphocytes was significantly longer than that of C and D lymphocytes. A lymphocytes did not label sufficiently to measure T _s. C and D lymph node lymphocytes and lymphocytes in tissues containing Reed-Sternberg cells had a longer T _s than splenic C and D cells. the former was: B , 12.7; C , 11.7; D , 11.0; combined, 11.8. the latter was: B , 12.2; C , 11.3; D , 11.2; combined, 11.6. the T _s of Reed-Sternberg cells in one specimen of a splenic Hodgkin's tumor was 13.1 hr. Macrophage T _s was 10.7 and 15.1 hr. Lymphosarcoma cell T _s was 14.2 and 14.6 hr. Reticulum cell sarcoma cell T _s was 7.5 and 7.7 hr.
The following minimum times were calculated from observation of ³H-TdR only labeled mitotic figures: S to prophase, 71 min; S to metaphase, 75 min; S to telophase, 100 min.     

Effect of serotonin depletion by p-chlorophenylalanine on serum prolactin levels in estrogen-treated ovariectomized rats: insights concerning the serotoninergic, dopaminergic and opioid systems.

The stimulatory effect of serotonin on prolactin secretion is well documented, and the administration of an inhibitor of serotonin synthesis (p-chlorophenylalanine - pCPA) has the expected inhibitory action on prolactin release in most experimental situations. However, there is evidence that in certain physiological or experimental conditions, activation of the serotoninergic system can also determine inhibition of prolactin secretion. The aim of the present study was to investigate the ability of estrogen to modify the effect of pCPA on prolactin secretion and to evaluate the participation of opioid and/or dopaminergic systems in regulating pCPA-induced prolactin secretion in estradiol-treated rats. We observed that pCPA administration (200 mg/kg/day, s.c., 2 days) to ovariectomized (OVX) female rats treated with estradiol benzoate (300 microg/week for 2 weeks, or 50 microg/week for 4 weeks, s.c.) causes a significant increase in serum prolactin, whereas no effect is observed in intact rats or in OVX rats without treatment. Bromocriptine administration completely reversed prolactin values previously increased by estradiol and by pCPA [OVX rats + estradiol = 86.50 ng/ml (68.90-175.02), OVX + estradiol + pCPA = 211.30 ng/ml (142.03-311.00), OVX + estradiol + pCPA + bromocriptine = 29.35 ng/ml (23.01 - 48.74), p<0.05. Naloxone administration partially reduced estrogen-induced high prolactin concentrations, but did not affect prolactin secretion stimulation determined by pCPA. Overall, the data from this report confirm the involvement of the dopaminergic system and, to a lesser degree, of endogenous opioids in prolactin secretion stimulation determined by estradiol. Furthermore, our results suggest that the stimulatory action of pCPA on prolactin secretion in estradiol-treated OVX rats is mediated by serotonin, which may also act indirectly on dopamine neurons.     

Prolactin receptor gene expression in rat splenocytes and thymocytes during oestrous cycle, pregnancy and lactation

Much evidence suggests that prolactin (PRL) has an immunoregulatory function. Part of this evidence is that the receptors for PRL are present on lymphocytes. Probably the effects of PRL on cells of the immune system depend on the level and specific forms of PRL-R present on the target cells. Therefore, PRL-R expression at both protein and mRNA levels was examined during oestrous cycle, pregnancy and lactation using Western blotting and PCR analysis. Antibody to the long form of PRL-R detected 84 and 42 kDa protein bands in the spleen but only 84 kDa band in the thymus. The expression pattern of these two protein bands was different in the spleen, suggesting that these two isoforms of PRL-R long form are differentially regulated by the hormones of oestrous cycle. In addition, depending on the tissue, the level of mRNA for the short and long forms of PRL-R showed a significant change at different stages of oestrous cycle. Moreover, 42 and 84 kDa PRL-R bands were detected in both spleen and thymus throughout the pregnancy and lactation; however, the expression pattern of 84 kDa protein band was different between tissues. This finding suggests that each tissue exhibits differential response to hormones which affect PRL-R content.     

Estrogen decreases the sensitivity of anterior pituitary to the inhibitory effect of nitric oxide on prolactin release

OBJECTIVE: We investigated the effect of chronic estrogen treatment on the inhibitory action of nitric oxide (NO) on prolactin release. METHODS: The effect of NO on prolactin release was studied in anterior pituitaries of female Wistar rats, intact at random stages, ovariectomized (OVX), and OVX treated for 15 days with 17beta-estradiol (OVX-E(2)). RESULTS: Sodium nitroprusside (NP, 0.5 mM), a NO donor, inhibited prolactin release from anterior pituitaries and was able to stimulate cGMP synthesis in intact and OVX rats. Only a high, supraphysiological concentration of NP (2 mM) inhibited prolactin release from anterior pituitaries of OVX-E(2) rats and increased cGMP synthesis in OVX-E(2) rats. 8-Br-cGMP, a cGMP analogue, decreased prolactin release from anterior pituitaries of OVX rats but did not affect it in OVX-E(2) rats. CONCLUSION: Our results suggest that estrogen may modify the sensitivity of the anterior pituitary to the inhibitory effect of NO on prolactin release by affecting guanylyl cyclase activity and the cGMP pathway.     

Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle

Abstract. Multivariate analysis of the expression of cyclin proteins and DNA content has opened new possibilities for the study of the cell cycle. By virtue of their cell cycle phase specificity, the expression of cyclins may serve, in addition to DNA content, as another marker of a cell's position in the cycle, and provide information about the proliferative potential of cell populations. Several applications of the methodology based on bivariate analysis of DNA content v . expression of B, E and D type cyclins are reviewed: 1 expression of cyclins by individual cells during their progression through the cycle can be studied, using exponentially growing cells without the necessity of cell synchronization or other perturbations of the cycle; 2 cells having the same DNA content but residing in different phases of the cycle (e.g. G₂ diploid v. G₁ tetraploid) can be distinguished; 3 cell transition from G₀ to G₁ and progression through G₁ (e.g. mitogen stimulated lymphocytes) can be assayed; 4 the population of proliferating cells can be distinguished from noncycling cells based on dual cell labelling with a G₁ and G₂ cyclin antibody; 5 cyclin restriction points can serve as additional cell cycle landmarks to map the point of action of antitumour drugs; 6 unscheduled expression of cyclins (e.g. the presence of cyclin B1 during G₁ and S) can be detected in several tumour transformed cell lines, possibly indicating disregulation of the machmery of cell cycle progression. The last finding 6 is of special importance, because such disregulation may be of prognostic consequence in human tumours.     

Prolactin mediates photoperiodic immune enhancement: effects of administration of exogenous prolactin on circulating concentrations, receptor expression, and immune function in steers

Changes in photoperiod can significantly impact the physiology of many species. For example, we have observed an improvement in cellular immune function in cattle on short-day photoperiod (SDPP) relative to long-day photoperiod (LDPP). In addition, prolactin (PRL) and PRL receptor (PRL-R) are affected by photoperiod management. Our hypothesis is that the inverse relationship observed between PRL and PRL-R mRNA expression during photoperiod treatment alters the sensitivity of the animal to PRL, thereby affecting the changes in their cellular immune function. The objective of this study was to determine the effects of exogenous PRL on photoperiodic-mediated immune responses. Eight Holstein steers received each of four treatments: LDPP (16L:8D), SDPP (8L:D), SDom (SDPP plus PRL via osmotic minipump for 10 days), and SDinj (SDPP plus PRL via 3x daily injections for 10 days). Steers on SDPP had decreased PRL relative to the other treatments. Expression of PRL-R mRNA was increased in SDPP animals relative to LDPP, SDom, and SDinj. Prior to PRL treatment, SDPP animals had greater lymphocyte proliferation and neutrophil chemotaxis relative to LDPP animals. Following PRL treatment, cellular immune function of SDom and SDinj animals was reduced to the level of LDPP animals. Addition of PRL to the in vitro lymphocyte proliferation did not alter response of LDPP animals but increased proliferation of lymphocytes from SDPP animals. The results of these experiments suggest that an animal's responsiveness to PRL correlate to changes in cellular immune function that occur with photoperiod manipulation.     

Biological and clinical aspects of prolactin receptors (PRL-R) in human breast cancer

PRL has a definite activity in the induction and promotion of mouse and in the growth of rat mammary tumors. We and others have found that human PRL or growth hormone (GH) had a growth promoting effect on human mammary cancer cells. It has been shown that prolactin receptors (PRL-R) which are specific for all lactogenic hormones (hPRL, hGH, hPL) are present on mammary cancer cells in long-term tissue culture and also in tumor biopsies. We found that 43% of the tumors had free PRL-R (FPRL-R) and that 72% had total PRL-R (TPRL-R) which have been desaturated in vitro. A significant correlation (Spearman test) was found between PRL-R (especially TPRL-R) on the one hand, estradiol (P<0.001) and progesterone receptors (P<0.01) on the other. The demonstration of PRL-induced proteins (PIP) might be a better sign of PRL sensitivity than the existence or PRL-R; PIP have been found by Northern blot analysis in 47% of 70 breast cancers. Overall survival (OS) and relapse-free survival (RFS) analysis with a median duration of follow-up of 5.3 yr showed that TPRL-R had a significant prognostic value only in node positive patients (χ²=5.61, P=0.02). Neither FPRL-R or TPRL-R were a significant prognostic factor when studied by Cox analysis. This confirms our previous results. Since at least some human mammary cancers appear to be PRL-dependent we carried out a multicenter randomized trial comparing as the first hormonal treatment tamoxifen (TAM) (30 mg/day)+bromocriptin (B) (5 mg/day) vs TAM + placebo. 171 patients entered this trial. No difference was observed between the two groups in response rates, duration of response or survival.

Recent studies are thus in favor of a role of lactogenic hormones during the course of breast cancer. However no improvement in therapy has been observed yet. The combination of drugs to achieve a total anti-lactogenic treatment, the use of anti-PRL-R antibodies are interesting areas of research; the recent cloning of PRL-R and GH receptors may open new clinical perspectives.     

Zinc binding to the gills of rainbow trout: the effect of long-term exposure to sublethal zinc

The effects of sublethal waterborne Zn (2·28 μmol l⁻¹) on Zn binding kinetics to the apical gill surface were studied in juvenile rainbow trout ( Oncorhynchus mykiss ). Two separate radiotracer techniques were employed to ascertain this information. First, in vitro binding kinetic experiments were performed at extremely elevated zinc concentrations (up to 20 mmol l⁻¹) to measure relatively low-affinity binding sites at the gill epithelium. There were no differences in Zn binding parameters ( K_m and B _max) for fish sublethally exposed to Zn for 21 days and their simultaneous controls. Nevertheless, Ca did have an increased inhibitory effect on Zn binding in Zn-exposed fish suggesting that the anionic groups on the gill epithelium of these fish had been altered in some manner. Additionally, in vivo Zn binding kinetics were investigated using environmentally relevant waterborne Zn concentrations (low μmol l⁻¹ range) to isolate high-affinity Zn binding sites (Ca transporters). No appreciable alterations in the Km and B _max values for Zn binding were seen between the Zn-exposed group and its simultaneous control following 15 days of exposure. Furthermore, no significant differences in CC morphometry were observed between treatments. Despite these lack of treatment effects, there were temporal alterations in K_m , B _max and CC fractional surface area in both groups. It is proposed that these fluctuations are controlled by hormonal factors (such as stanniocalcin), believed to play a role in Ca influx.     

雌激素对SD大鼠肾脏缺血再灌注肾小管上皮细胞Cx43蛋白表达的影响

为了探讨雌激素（estrogen,E2）对SD大鼠肾脏缺血再灌注（ischemia-reperfusion,I/R）肾小管上皮细胞Cx43蛋白表达的影响,选取32只健康雌性SD大鼠作为实验材料,所有雌性SD大鼠均先实施去卵巢（ovariectomized,OVX）处理,之后32只雌性OVX SD大鼠随机分为OVX组、OVX+假手术组、OVX+I/R组、OVX+I/R+E2组,每组8只。去卵巢2周后,OVX组不进行任何处理,OVX+Sham组进行假手术处理,OVX+I/R组进行缺血再灌注处理,OVX+I/R+E2组在进行缺血再灌注处理前,连续3 d予以雌激素处理。通过比较各组SD大鼠血肌酐水平、尿素氮水平、肾脏HE染色及Paller评分,评价肾脏组织损伤程度,并通过免疫组化和蛋白印迹法分析各组SD大鼠肾小管上皮细胞中Cx43蛋白的表达位置及水平。与OVX组比较,OVX+I/R组血清BUN、SCr水平明显升高（P<0.05）;与OVX+I/R组比较,OVX+I/R+E2组血清BUN、SCr水平明显降低（P<0.05）。HE染色观察发现,与OVX组相比,OVX+I/R组肾小管扩张...     

A Prolactin Action on Acetylcholine Metabolism in Striatum, Hippocampus, and Thalamus

Abstract: Since prolactin can regulate the release of striatal dopamine, we have evaluated the functional implications of this effect by studying the action of injected prolactin on the turnover rate of acetylcholine (TR_ACh) in various brain areas. We selected striatum and hippocampus as two areas in which dopaminergic terminals are known to regulate TR_ACh and frontal and parietal cortex as areas where dopamine has little or no control on TR_Ach. Intraventricularly injected prolactin reduces the TR_ACh in striatum, hippocampus, and thalamus but not in the two cortical areas. Intraseptal injection of prolactin reduces TR_ACh in hippocampus, suggesting that this polypeptide acts on hippocampus by changing the activity of afferent neurons impinging upon the cell bodies of the cholinergic septal-hippocampal neurons. The reductions in TR_ACh induced by intraventricular prolactin in hippocampus and striatum are nullified by 6-hydroxydopamine-induced lesions of dopaminergic neurons located in areas A₉ and A₁₀. These results suggest that the presence of dopaminergic neurons is required to obtain the prolactin-elicited decrease of TR_Ach.     

INHIBITION OF THYROXINE-INDUCED RESORPTION OF TADPOLE TAIL BY ADENOSINE 3', 5'-CYCLIC MONOPHOSPHATE

Small segments of tail of Bufo bufo japonicus tadpoles were cultured in medium containing thyroxine (T₄) and dibutyryl cyclic AMP (dbcAMP). Like prolactin, the cyclic nucleotide blocked T₄-induced shrinkage or tail pieces. Histological study of the segments after 4-days culture revealed that dbcAMP suppressed degenerative changes induced by T₄. The inhibitory effect of prolactin on T₄-induced tail regression was promoted by caffeine, an inhibitor of adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-phosphodiesterase.
The effect of prolactin on the level of cyclic AMP in the tail was also studied in vivo . Sixty min after prolactin injection, the cyclic AMP level was 2–3 times the control value. Possible involvement of cyclic AMP in the action of prolactin, which blocks tail resorption induced by T₄, was discussed.     

Modulation of the rats' immune status by monoassociation with anaerobic bacteria

The capacity of a pure culture of anaerobic intestinal bacteria to influence the host's cellular and humoral immune systems was investigated with germfree, monoassociated, and conventionally reared rats. Monoassociation of germfree rats with Bacteroides fragilis stimulated the production of serum gamma globulin, agglutinating antibodies, and an apparent IgG (immunoelectrophoresis) band. A comparison of the in vitro blastogenic potential of lymphocytes (spleen cells and mesenteric lymph node cells) from germfree, monoassociated, and conventionally reared rats indicated the following: (1) the microbial flora had no obvious effect on the capacity of nonstimulated lymphocytes to incorporate [3H]thymidine; (2) spleen cells from conventionally reared rats responded to phytohemagglutinin, concanavalin A, or pokeweed mitogen better than splenocytes from germfree rats; (3) colonization of germfree rats with Fusobacterium necrophorum increased the responsiveness of splenocytes to photohemagglutinin and concanavalin A; and (4) monoassociation of germfree rats with B. fragilis, but not with F. necrophorum or propionibacterium acnes, increased splenocyte blastogenesis to homologous (i.e., colonizing) bacterial antigens. This study indicated that some intestinal bacteria can modulate the immune status of the host; the extent and nature of this modulation depended on the particular species of colonizing bacteria.