Immunosuppressive and graft-rejecting anti--bodies in heterologous anti-lymphocytic serum.

Skin allografts survived longer on ALS-treated, complement-deficient (C5 negative) recipients than on ALS-treated, complement-competent (C5 positive) recipients. Administration of C5-positive serum to C5-negative, ALS-treated recipients resulted in reduced graft survival. A percentage of grafts from ALS-treated, C5-positive donors was rejected when transferred to untreated syngeneic recipients; this was not observed when C5-negative, syngeneic animals served as ALS-treated donors and untreated recipients. It was concluded that ALS has graft-rejecting properties which are promoted by late acting complement components. Unlike ALS-mediated graft rejection, ALS-mediated immunosuppression appeared to be independent of the late acting complement components. The effect of ALS on the humoral response to sheep erythrocytes was examined in complement-deficient and complement-competent mice. Immune-suppression was determined by ALS treatment of C5-competent and C5-deficient mice and also by transfer of in vitro ALS-treated spleen cells from C5-negative and C5-positive donors to cyclophosphamide-treated recipients. The ability of ALS to depress the humoral response to sheep cells and to decrease immunological competence of spleen cells was the same in the presence as in the absence of C5.

Tumor colony formation by Friend virus-infected cells in immunosuppressed mice.

We have investigated the role of host immunological factors in the formation of "tumor colonies" in the spleens of unirradiated C57BL/6 X C3Hf/Bi FI mice 9 days after i.v. injection of spleen cells from Friend virus (FV)-infected C3Hf/Bi donors. Pretreatment of hosts with antilymphocyte serum (ATS) increased the number of tumor colonies. Pretreatment with formalinized FV-infected cells had the opposite effect, and ATS diminished the inhibitory effect of preimmunization. Cell suspensions from 11 individual FV-infected donors were examined. The suspensions differed with respect to their behavior on transplantation into untreated and ATS-pretreated F1 hybrid hosts. With several suspensions, the number of tumor colonies produced was approximately proportional to the number of cells injected; in all of these, ATS increased the slope of the line relating colony number to cell number. With most of the suspensions, tumor colony-forming efficiencies in untreated hosts strikingly decreased with increasing number of cells injected; ATS induced an increase in the number of tumor colonies and rendered the colony-forming response more nearly proportional to cell number. With two suspensions, few or no colonies developed; pretreatment with ATS had no significant effect. When the 11 cell suspensions were considered together, a proportional relation was found between the magnitude of the ATS effect (i.e., colony number in the presence of ATS minus colony number in the absence of ATS) and the colony-forming efficiency in ATS-treated mice. The ATS effect on the average was equivalent to a 2-fold increase in tumor colony-forming efficiency. We interpret these findings to indicate that two factors interact to determine the number of tumor colonies produced by spleen cells from FV-infected C3H donors in untreated F1 hybrid hosts. One is a property of the FV-infected cell population and includes its frequency of tumor colony-forming units; this factor varies widely among different cell suspensions. The other is a property of the tumor colony-forming units-host interrelationship and includes the vulnerability of tumor colony-forming units to the host immune response elicited by the injected cells; this factor appears to be constant with different cell suspensions. The present results show that the two factors can be dissociated in immunosuppressed hosts.

Chick retinal pigment epithelium. A new culture system for studying H2-histamine receptors.

Histamine elevates the intracellular cyclic AMP levels in cultured embryonic chick retinal pigment epithelium. The half-maximal activity is 6 X 10(-6) M. The effect of histamine is mediated by H2 receptors, i.e., inhibited by the H2 antagonist cimetidine and not affected by the H1 antagonists diphenhydramine and pyrilamine. The inhibition constant (KI) of cimetidine is 1.3 X 10(-8) M. Thus, the present system offers the opportunity of studying the nature of coupling between histamine receptors and adenylate cyclase under controlled conditions in a homogeneous cell type.

Immunosuppression after injection of Friend virus into C57BL/6 mice of different ages.

Injection with Friend virus (FV) causes immunosuppression in young and old C57BL/6 mice, i.e. it occurs whether or not the virus replicates very briefly or for a long period. There are only minor age-related differences in the extent of immunosuppression, except that suppression appears to persist somewhat longer in old than in young animals.

Epinephrine regulates cholinergic transmission mediated by rat retinal neurons in culture.

The purpose of this study was to investigate the effects of epinephrine on neurotransmission mediated by cholinergic neurons derived from the rat retina. We used a culture system in which striated muscle cells served as postsynaptic targets for cholinergic neurons of the embryonic retina. This culture system permitted the physiological monitoring of acetylcholine released by retinal neurons. Here, we report that epinephrine facilitates evoked transmission across retina-muscle synapses. This facilitation of cholinergic transmission by epinephrine is reversible, can be mimicked by isoproterenol (a beta adrenoceptor agonist) and blocked by propranolol (a beta adrenoceptor antagonist). Neither the alpha-2 adrenoceptor blocker, yohimbine, nor the dopamine receptor antagonist, haloperidol, blocked this effect of epinephrine. Since epinephrine was found not to influence the membrane potential of muscle cells nor the responses of myotubes to acetylcholine, epinephrine appeared to have mediated its facilitatory effect on cholinergic transmission by affecting retinal cells. Because previous findings indicated that adenosine 3',5'-cyclic monophosphate may be involved in the modulation of transmission at retina-muscle synapses, the effect of epinephrine on adenosine 3',5'-cyclic monophosphate levels was investigated. Our biochemical studies demonstrated that epinephrine could increase adenosine 3',5'-cyclic monophosphate levels markedly in cultured retinal cells. The accumulation of adenosine 3',5'-cyclic monophosphate induced by epinephrine could be blocked by propranolol, but not by yohimbine nor haloperidol. Taken together, the results indicate that the facilitatory effect of epinephrine is mediated via a beta adrenoceptor and may involve an increase in adenosine 3',5'-cyclic monophosphate levels. Our findings are in agreement with the hypothesis that epinephrine may be a modulatory neurotransmitter in the rat retina.

Corneal endothelial cell survival in organ cultures under acute oxidative stress: effect of VIP.

PURPOSE: Human corneal endothelium, a neural crest-derived tissue, has a very limited regenerative capacity and may depend on trophic factors for its survival throughout life, as well as after injury and during storage before transplantation. The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a 28-amino acid neurotrophic factor present in human aqueous humor, promotes the survival of corneal endothelium in corneal organ cultures, and whether VIP is produced by the corneal endothelium. METHODS: Thirteen viable human donor corneas that had been received from the Central Florida Lions Eye Bank and stored in preservation medium (Optisol-GS; Chiron Vision, Irvine, CA) at 4 degrees C for 8 to 17 days were bisected. Each half was treated with either 0 or 10 nM VIP (15 minutes) and subjected to H(2)O(2) (1.4 mM, 30 minutes) treatment at 37 degrees C. The numbers of live and dead corneal endothelial (CE) cells isolated from the corneas were then determined under fluorescence microscopy using a live-dead viability-cytotoxicity assay conducted by an observer uninformed of the treatment. The effect of VIP (10(-16) to 10(-6) M) on CE cell survival was also studied in fresh bovine corneas in situ, by using the same assay. The presence of VIP in the corneal endothelium in fresh human donor and bovine eyes was examined by immunocytochemistry, in situ hybridization, and Western blot analysis, whereas VIP in the bovine aqueous humor was assessed by radioimmunoassay. RESULTS: VIP (10 nM) significantly increased CE survival in 10 of 13 human corneas. The mean survival of CE cells (+/-SEM) was 42% +/- 3% in control corneas versus 59% +/- 3% in VIP-treated corneas (P < 0.001). In bovine corneas, VIP at concentrations as low as 10(-10) M demonstrated a significant protective effect. The mean number of dead CE cells on bovine corneas was maximally decreased by 10(-6) M VIP from 46 +/- 5 to 18 +/- 3 per field. In CE cells from fresh human and bovine corneas, VIP immunoreactivity and mRNA were detected. VIP was also present in bovine aqueous humor at 40 +/- 8 pM. CONCLUSION: VIP may be an autocrine trophic factor that protects CE cells from H(2)O(2) in normal aqueous humor and possibly from other oxidative insults.

Vasoactive intestinal peptide stimulation of human trabecular meshwork cell growth.

PURPOSE: To demonstrate that vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, is a growth factor of human trabecular meshwork (TM) cells in culture and in a corneoscleral explant organ culture treated with laser trabeculoplasty (LTP). METHODS: Proliferating human TM cells in cell cultures were incubated with VIP for 20 hours, followed by total cell number determination, using a Coulter counter. The percentage of proliferating TM cells was assessed, using an antibody against the proliferating cell nuclear antigen (PCNA). To test the growth effect of VIP on TM cells in situ, corneoscleral explants in organ cultures were first treated with argon LTP to initiate TM-cell proliferation and then were exposed to VIP for 48 hours. The mitotic TM cells were demonstrated immunocytochemically, using anti-PCNA in paraffin sections of the explants; and the total number of TM cells was determined after paraffin sections were counterstained by hematoxylin. RESULTS: Vasoactive intestinal peptide dose-dependently stimulated the proliferation of TM cells in cell culture. Treatment with 5 x 10(-10) M VIP resulted in a maximal increase of 40% in cell number. The effect of VIP was blocked by a VIP antagonist. The number of PCNA-stained TM cells and the total cell number in the TM in LTP-treated corneoscleral explants were increased by VIP. CONCLUSIONS: Exogenously applied VIP stimulated the proliferation of human TM cells in subconfluent cultures and in LTP-treated corneoscleral explants. In that LTP has been shown to increase the number of TM cells in situ, the growth stimulatory effect of VIP may help enhance this therapy.

Age-dependent separation of class-specific suppressor cells in thymus of SJL/J mice.

The thymus of SJL/J mice of age 3-6 weeks has been previously shown to contain suppressor cells that inhibit the antibody response to lymph node cells to SRBC. The effect of these suppressor cells disappear as the animals age (24 weeks or more). We find that these aged animals acquire thymic suppressor cells which suppress the generation of cytotoxic T-cells both in vitro and in vivo. Although such suppressors are not present in the thymuses of young SJL/J mice, suppression can be induced by treatment with estrogen and progesterone. The differentiation of functionally different suppressor cell populations in thymus may be affected by both age and hormonal status.

Dietary fat alters progression of some age-related changes of the immune system.

SJL mice develop resistance against tolerance between the 9th and 25th wk of life. This resistance is linked with a loss of suppressor capacity in the thymus. We have shown here that contact photosensitivity (CPS) decreases as a function of age and that this is due to an age-dependent increase in suppressor capacity. Diet fats have a differential effect on age-dependent changes in suppressor activity; a low P/S diet prevents or delays loss of suppressor activity for antibody formation and a high P/S diet prevents or delays the development of suppressor activity in CPS reactions.

Lymphokine-activated killer (LAK) cells: I. Age-dependent decline of LAK cell-mediated cytotoxicity.

Experiments were designed to assess age-related changes in generation of lymphokine-activated killer (LAK) cells and to test whether these changes can be modified by diets differing in the proportion of polyunsaturated to saturated fatty acids (P/S). Ficoll-Hypaque-isolated spleen lymphocytes of rodent chow-fed, 6-85-week-old C57BL/6 (H-2b), 8-81-week-old C57BL/10 (H-2b) and 6-62-week-old SJL (H-2s) mice were cultured in IL-2-containing medium and examined in 51Cr cytotoxicity assay. Similarly, Ficoll-Hypaque-isolated spleen lymphocytes of 6-36-week-old SJL mice fed diets which differed in the ratio of polyunsaturated/saturated fatty acids were cultured in IL-2-containing medium and assayed for cytotoxicity. Age-related decline of LAK cell-mediated cytolysis was observed in mice of both H-2b and H-2s haplotype. The age-related decline of LAK cell-mediated cytolysis was the consequence of age-related decrease in the rate of LAK cell precursor maturation. SJL mice fed from birth with diets differing in P/S did not differ in LAK cell-mediated cytolysis.

Modulation of a 190-kD microtubule-associated protein in pigment epithelium by VIP.

Vasoactive intestinal peptide stimulates phosphorylation of six high molecular weight cytosolic proteins in the cultured retinal pigment epithelium (RPE). Of these, the 190-kD phosphoprotein is associated with the microtubules assembled by taxol/GTP and is immunologically related to the brain microtubule-associated protein 2 (mol.wt. = 280 kD). VIP is also shown here to stimulate secretion in the cultured RPE. VIP-stimulated phosphorylation of a 190-kD microtubule-associated protein is also demonstrated here in the retinal glia.

Bombesin activates MAP kinase in non-small cell lung cancer cells.

The effects of bombesin (BB) on mitogen activated protein (MAP) kinase were investigated using non-small cell lung cancer (NSCLC) cells. By Western blot, both 42 and 44 kDalton forms of MAP kinase were present in NCI-H1299 and NCI-H838 cells. Addition of BB to NCI-H1299 cells resulted in phosphorylation of the MAP kinase substrate myelin basic protein (MBP). Phosphorylation of MBP was maximal 6 min after the addition of 10 nM BB to NCI-H1299 cells. Addition of gastrin releasing peptide (GRP) or GRP14-27 but not GRP1-16 to NCI-H 1299 cells caused MBP phosphorylation. The effects of BB were inhibited by BW2258U89, a BB receptor antagonist, and PD98059, a MAP kinase kinase inhibitor. Also, PD98059 inhibited the clonal growth of NCI-H1299 cells. These data suggest that MAP kinase may be an important regulatory enzyme in NSCLC.

Frequency of spinocerebellar ataxia types 1,2,3,6,7 and dentatorubral pallidoluysian atrophy mutations in Korean patients with spinocerebellar ataxia.

Autosomal dominant cerebellar ataxias (ADCAs) are a heterogeneous group of disorders characterized by degenerative symptoms in the cerebellum, spinal cord, and brain stem. Six different genes have been reported to be associated with ADCA, and the length of trinucleotide repeats of these genes is correlated with the age at onset and severity of symptoms. Although there are strong hereditary effects in these disorders, most of the studies carried out in heterogeneous populations and in small groups obscure the true incidence of these diseases. We examined the frequency of six types of ADCAs in 87 unrelated Korean patients with progressive ataxia and compared the results to the frequencies in other ethnic groups. Spinocerebellar ataxia (SCA) type 2 was the most frequent hereditary ataxia (12.6%) and types 3 and 6 accounted for 4.6% and 6.9% of ataxia patients, respectively. Dentatorubral pallidoluysian atrophy was also found in three patients (3.4%). No instances of SCA types 1 or 7 were detected. These findings show the striking contrast to the white population and a difference from Japanese findings. Our results demonstrate that dentatorubral pallidoluysian atrophy should be included in the differential diagnosis of Korean patients with spinocerebellar ataxia, and that there are strong hereditary effects in patients with ADCAs.

Effects of agonists on the intracellular cyclic AMP concentration in monkey trabecular meshwork cells.

We examined the effects of several neuroendocrine agents on the intracellular cyclic AMP level in cultured monkey trabecular meshwork cells. Among the peptide agonists studied, only the vasoactive intestinal peptide elevated the cyclic AMP level. Alpha-melanocyte stimulating hormone, glucagon, and others showed no stimulation. Prostaglandin E1 and L-isoproterenol also enhanced the intracellular cyclic AMP level. Such effects may implicate physiologic roles of these agonists in the trabecular meshwork.

VIP modulation of cultured glial cells of the rat retina.

Cultured retinal glial cells from the rat are responsive to modulation by vasoactive intestinal peptide (VIP). VIP (1 X 10(-6) M) elevated the intracellular cyclic AMP concentration from the basal level of (4.4-11.1) p mole/mg protein to (354-440) p mole/mg protein in three minutes at 25 degrees C. The half-maximal concentration is 4.8 X 10(-8) M, which is similar to that observed in the cultured retinal glial cells from the chick embryo.

Modulators of cyclic AMP in monolayer cultures of Y-79 retinoblastoma cells: partial characterization of the response with VIP and glucagon.

VIP markedly stimulates intracellular cAMP accumulation in the human retinoblastoma Y-79 cell line. cAMP increased about 5-fold above the basal level with 10(-8)M VIP and reached a maximum level (about 70-fold increase) with 2 X 10(-6)M VIP. Glucagon at 6 X 10(-8)M significantly increased cAMP accumulation with a maximal response at 4 X 10(-7)M. Secretin was only effective at micromolar concentrations. Glucagon at 2 X 10(-6)M had a synergistic effect with VIP at 2 X 10(-8)M. Of other substances tested, L-isoproterenol (25-fold increase) and PGE1 (4-fold increase) were most effective. These results demonstrate that VIP and glucagon modulate cAMP accumulation in Y-79 cells and provide a model for studying the effect of these substances on function of neuronal and on malignant cells in vitro.

Evidence of a functional VIP receptor in cultured human retinal pigment epithelium.

The effect of VIP on the intracellular cyclic AMP of human retinal pigment epithelium cultures has been studied. Functional VIP receptor has been demonstrated in cultures from eyes given by five normal donors (age 16-64) (N-HRPE). But it has been found to be absent from high passage number cultures obtained from a retinitis pigmentosa eye of an 84-year-old patient (RP-HRPE). After 3 min of reaction with 1 x 10(-6) M VIP, the intracellular cyclic AMP level has increased to 5-15-fold over the basal level. The maximal effect of VIP (20-fold over the basal level) has been observed at 1 x 10(-7) M VIP. The half maximal activity of VIP is 3-5 x 10(-8) M. The present study also demonstrates the inducibility of the VIP responsiveness in RP-HRPE cultures after they have been treated with butyrate. Curr. Eye Res. 14: 1009-1014, 1995.

VIP stimulation of polarized macromolecule secretion in cultured chick embryonic retinal pigment epithelium.

Vasoactive intestinal peptide (VIP) stimulated macromolecule secretion at the apical membranes of the chick embryonic retinal pigment epithelium cultured on permeable supports in a time- and concentration-dependent manner. VIP stimulated secretion of molecules with MW of 80, 74, 70, 60, 42, 35, 24, 20, and 14 kDa. A 1.9- to 2.6-fold stimulation in secretion of molecules with MW greater than 10 kDa precipitable by 10% trichloroacetic acid was observed after treatment with 1 microM VIP for 15 min. The effect of 1 microM VIP was mimicked by 10 microM dibutyryl cyclic AMP and attenuated by dopamine (1 x 10(-4) M), while colchicine, beta-lumicolchicine, and monensin, all at 1 microM, had no effect.

Signal transduction through the vasoactive intestinal peptide receptor stimulates phosphorylation of the tyrosine kinase pp60c-src.

The present study demonstrates that signal transduction through a receptor lacking intrinsic tyrosine protein kinase activity involves a rapid and potent phosphorylation of a non-receptor tyrosine protein kinase in the membranes. Vasoactive intestinal peptide (VIP) stimulates phosphorylation of a membrane protein with a M.W. of 56 KD (pp60) in the cultured chick embryonic retinal pigment epithelium. VIP stimulates phosphorylation of the pp60 with such efficiency and potency that the maximal phosphorylation has been observed at the earliest time (3 minutes at 1 x 10(-6)M VIP) and the lowest concentration (1 x 10(-11)M for 20 minutes) examined. Western blot analysis with a monoclonal antibody anti-pp60src (GD11, Parsons et al., J. Virol. 51, 272-282, 1984) indicates that the pp60 is the pp60c-src, a normal cell oncogene product with intrinsic tyrosine protein kinase activity.

The chick retinal pigment epithelium grown on permeable support demonstrates functional polarity.

The retinal pigment epithelium (RPE) from the chick embryo was cultured on permeable support. Using confluent cultures and analysis of the incubation medium, the present study demonstrates that RPE cells cultured on permeable membrane retain functional polarity, a characteristic of the RPE in vivo. The degree of intercellular permeability in the confluent RPE cultures was estimated by following [3H]inulin movement from the apical side to the basal side of the cultures. Twenty-four hours after exposure of the apical side of the culture to [3H]inulin, the 3H concentration in the apical medium remained at 3.4 to 4.4 times of that in the basal medium. The barrier function of RPE disappears in the presence of EDTA. Net unidirectional fluid movement from the apical side of the cultures to the basal side of the cultures is regularly observed in confluent RPE cultures. The rate varies among different preparations of cultures and the highest is 1.60-1.84 microliters/cm2/h. When cultures are given 26 h of [35S]methionine, more than 20 bands with molecular weights ranging from 20,000 to greater than 250,000 Da can be detected in the medium as assessed by autoradiography of SDS-polyacrylamide gels. While six macromolecules appear to be equally concentrated in the basal medium and the apical medium, the majority are in higher concentration in the basal medium. Analysis of the 10% TCA-precipitable fraction of the medium showed that the specific activities in the apical medium and basal medium were 24.0 +/- 0.4 X 10(6) and 46.4 +/- 0.2 X 10(6) (mean +/- SEM, N = 8) cpm/ml/mg RPE protein, respectively. When cultures react with VIP (vasoactive intestinal peptide), the elevated intracellular cyclic AMP is extruded into the medium bathing the cells. However, the rate of extrusion into the basal medium is twice as fast as that into the apical medium. Electron microscopy of the confluent RPE cultures shows morphological polarization of the cells. The intercellular spaces appear to be closed at the apical side of the cells by junctional complexes consisting of tight junctions, zonular adherens junctions, and gap junctions.