Beta-endorphin neuronal cell transplant reduces corticotropin releasing hormone hyperresponse to lipopolysaccharide and eliminates natural killer cell functional deficiencies in fetal alcohol exposed rats

Abstract

Background: Natural killer (NK) cell dysfunction is associated with hyperresponse of corticotropin releasing hormone (CRH) to immune challenge and with a loss of beta-endorphin (BEP) neurons in fetal alcohol exposed animals. Recently, we established a method to differentiate neural stem cells into BEP neurons using cyclic adenosine monophosphate (cAMP)-elevating agents in cultures. Hence, we determined whether in vitro differentiated BEP neurons could be used for reversing the compromised stress response and immune function in fetal alcohol exposed rats.

Methods: To determine the effect of BEP neuron transplants on NK cell function, we implanted in vitro differentiated BEP neurons into the paraventricular nucleus of pubertal and adult male rats exposed to ethanol or control in utero. The functionality of transplanted BEP neurons was determined by measuring proopiomelanocortin (POMC) gene expression in these cells and their effects on CRH gene expression under basal and after lipopolysaccaride (LPS) challenge. In addition, the effectiveness of BEP neurons in activating NK cell functions is determined by measuring NK cell cytolytic activity and interferon-gamma (IFN-gamma) production in the spleen and in the peripheral blood mononuclear cell (PBMC) following cell transplantation.

Results: We showed here that when these in vitro differentiated BEP neurons were transplanted into the hypothalamus, they maintain biological functions by producing POMC and reducing the CRH neuronal response to the LPS challenge. BEP neuronal transplants significantly increased NK cell cytolytic activity in the spleen and in the PBMC and increased plasma levels of IFN-gamma in control and fetal alcohol exposed rats.

Conclusions: These data further establish the BEP neuronal regulatory role in the control of CRH and NK cell cytolytic function and identify a possible novel therapy to treat stress hyperresponse and immune deficiency in fetal alcohol exposed subjects.

Fig. 1

Immunocytochemical characterization of neural stem cells before and after differentiation. Neuronal stem cells were stained for Nestin (left) and Vimentin (middle) prior to differentiation and stained for BEP (right) after differentiation. “-”, 10 mm.

Fig. 2

Physiological responses of transplanted cells in the PVN. Male rats (35- to 40-day old) fed during embryonic days 11 through 21 via dams; with alcohol (alcohol-fed rats; AF) or isocaloric liquid diet (pair-fed rats; PF) were transplanted with BEP cells (20,000 cells/1 μl) into the left PVN or nonviable BEP cells (CONT; 20,000 cells/1 μl) into the right PVN. After 2 weeks, rats underwent LPS or saline treatment and then 3 hours after they were sacrificed and the PVN tissues of these rats were collected and used for POMC or CRH measurements. POMC mRNA (A) and CRH mRNA (B) levels in the PVN lobe with BEP cells and in the contralateral lobe of the PVN with CONT after i.p. administration of LPS or of saline. n =6. POMC mRNA data identified no interaction between in utero feedings and cell treatments (F = 0.5116, df-3, p < 0.6781), while CRH mRNA data showed significant interaction between in utero feedings and cell treatments (F = 161.1, df-3, p < 0.001), ^ap < 0.001 versus PF + CONT + saline or AF + CONT + saline. ^bp <0.01 versus PF + CONT + LPS or AF + CONT + LPS. ^cp < 0.01 versus PF + BEP + saline. ^dp < 0.001 versus rest of the groups.

Fig. 3

Determination of the effect of BEP transplants in the PVN on NK cell cytolytic function. Adult male rats (90-day old) fed during embryonic days 7 through 21 via dams; with alcohol (alcohol-fed rats; AF), an isocaloric liquid diet (pair-fed rats; PF) or a regular diet (ad lib-fed rats; AdF) were transplanted with BEP (20,000 cells/1 μl) or cortical cells (CORT; 20,000 cells/1 μl) into the left PVN. After 4 weeks, rats were sacrificed and the spleens were collected. Splenocytes were prepared and assayed for NK cell cytolytic activity against YAC-lymphoma cells by a standard 4 hours ⁵¹Cr release cytolytic assay. The histograms represent mean ± SEM of NK activity in lytic units (LU). n = 5 to 7 rats. NK cytolytic activity data identified significant interaction between in utero feedings and cell treatments (F = 10.66, df-4, p < 0.001). ^ap < 0.001 versus AdF and PF rats. ^bp < 0.001 versus CORT cell-treated or untreated animals fed similarly during embryonic life.

Fig. 4

Comparison of the effect of unilateral or bilateral BEP transplants in the PVN on NK cell functions. Adult male rats (90-day old) alcohol-fed (AF) or pair-fed (PF) during the prenatal period were transplanted with BEP or cortical cells (CORT) into 1 PVN (+) or 2 PVN (++). After 3 weeks, the spleen and approximately 1 ml of blood was collected from the orbital sinus of each rat and used for peripheral blood mononuclear cell (PBMC) preparation and plasma separation. (A, B) PBMC and splenic samples were used to determine the NK cytolytic activity by a standard 4 hours ⁵¹Cr release cytolytic assay. (C) Plasma samples were used to determine IFN-γ. The histograms represent mean ± SEM values from 5 to 7 rats. NK cytolytic activity of PBMC and spleen data identified no significant interaction between in utero feedings and cell treatments (PBMC, F = 2.781, df-2, p < 0.083; Spleen, F = 0.8398, df-2, p < 0.4436). There was also no significant interaction between in utero feeding and cell treatment of the IFN-γ response to cell transplants (F = 0.1553; df-2; p < 2.029). ^ap < 0.05 versus CORT-cell transplant in rats that were similarly treated prenatally. ^bp < 0.01 versus +BEP-cell transplant in rats that were similarly treated prenatally. ^cp < 0.001, compared to CORT-cell transplants and PF-treated groups.