Lysis of human arterial smooth muscle cells infected with herpesviridae by peripheral blood mononuclear cells: implications for atherosclerosis.

The cytotoxicity of peripheral blood mononuclear cells (PBMC) to human arterial smooth muscle cells (SMC) infected with cytomegalovirus (CMV) or herpes simplex virus-1 (HSV) was investigated. PBMC were isolated from heparinized blood of healthy donors by Ficoll-Hypaque centrifugation and were tested for cytotoxicity against human SMC or human fibroblast-like (MRC-5) cells infected with CMV or HSV, using the chromium-51 (51Cr) release cytotoxicity assay. Both SMC and MRC-5 cells infected with either CMV (SMC-CMV), (MRC-5-CMV), or HSV (SMC-HSV), (MRC-5-HSV) were lysed by PBMC above background lysis of uninfected SMC cells. Treatment of PBMC with NK-specific monoclonal CD16 antibody and rabbit complement reduced greatly the lysis of SMC, SMC-CMV, and K562 cells, suggesting that lysis of different types of target cell by PBMC was mediated mainly by natural killer (NK) cells. The pattern of natural cytotoxicity against SMC-CMV was different from that against SMC-HSV. Maximum lysis of SMC-CMV was observed at 24 hr postinfection compared to 8 hr postinfection for SMC-HSV. NK reactivity against SMC-CMV increased from 8 to 24 hr postinfection, followed by a gradual decline at 48 and 72 hr. Supernatants generated by culturing SMC-CMV or coculturing SMC-CMV with PBMC enhanced NK cell-mediated lysis of SMC or SMC-CMV. Natural cytotoxic reactivities of PBMC against SMC-CMV or SMC-HSV may occur in vivo. Such reactions could moderate the interaction of these viruses with vascular SMC and could influence the development and/or the progression of atherosclerotic lesions.

Genetic resistance to lymphoma-leukemia: role of natural killer cells in the rejection of lymphoma grafts.

Lethally irradiated genetically resistant (C57 x AKR) mice, unlike non-resistant (C3H x AKR) mice, which display genetic resistance to transplantation of parental C57 bone marrow grafts (GR to BMT), also reject transplanted parental AKR lymphoma grafts (GR to lymphoma). Depletion of natural killer (NK) cell activity of resistant (C57 x AKR) mice by anti-asialo GM-1 serum abrogated the resistance, whereas stimulation of NK cell activity of non-resistant (C3H x AKR) mice by polyinosinic: polycytidillic acid (poly I:C) induced the resistance to parental AKR lymphoma grafts. This shows that NK cells contribute to GR of (C57 x AKR) hybrid mice to parental AKR lymphoma grafts. Further, these results indicate that NK cells which confer genetic resistance to bone marrow transplantation (GR to BMT) in lethally irradiated (C57 x AKR) hybrid mice, also mediate GR to lymphoma transplantation in these mice, thereby linking GR to BMT with GR to lymphoma-leukemia.

Developmental expression of adenosine deaminase in the upper alimentary tract of mice.

The distribution and localization of adenosine deaminase (ADA) was studied during postnatal development of the alimentary tract in mice. There was detectable enzyme activity in all organs examined, but a range of more than 10,000 fold in the relative levels of specific activity was observed among adult tissues. A comprehensive survey of multiple adult tissues revealed that the highest levels of ADA occur in the upper alimentary tract (tongue, esophagus, forestomach, proximal small intestine). Immunohistochemical analysis revealed that ADA was predominantly localized to the epithelial lining of the alimentary mucosa: the keratinized squamous epithelium that lines the forestomach, esophagus, and surface of the tongue; and the simple columnar epithelium of the proximal small intestine (duodenum, proximal jejunum). Biochemical analysis revealed that ADA was one of the most abundant proteins of these mucosal tissue layers, accounting for 5%-20% of the total soluble protein. Tissue-specific differences in ADA activity correlated both with levels of immunoreactive protein and RNA abundance. The level of ADA activity in the upper alimentary tissues was subject to pronounced developmental control, being low at birth and achieving very high levels within the first few weeks of postnatal life. The appearance in development of ADA-immunoreactivity coincided with maturation of the mucosal epithelium. These results suggest that ADA is subject to strong cell-specific developmental regulation during functional differentiation of certain foregut derivatives in mice.

Expression of human argininosuccinate synthetase in murine hematopoietic cells in vivo.

As part of an effort to develop a model system for somatic gene therapy, a human argininosuccinate synthetase (AS) cDNA has been introduced into two gag+ retroviral vectors, and high titer clones were obtained for both constructs. The presence of proviral DNA sequences was detected in individual spleen colonies after infection of primary murine marrow cells with each virus. Mice were reconstituted for long-term survival with marrow infected with one virus, and they demonstrated elevated levels of AS expression in peripheral blood for up to eight weeks posttransplantation.

Role of NK cells in resistance of Syrian golden hamsters to radiation-induced lymphoma-leukemia.

The results suggest that hamsters are more resistant than mice to radiation leukemogenesis because their NK cell activity recovers more quickly after fractionated irradiation without marrow injection. This correlates well with, and further corroborates, the important role of NK cell activity in the prevention of irradiation leukemogenesis.

Replication of cytomegalovirus in human arterial smooth muscle cells.

Cytomegalovirus (CMV) strain AD-169 replicated in smooth muscle cell (SMC) cultures derived from human umbilical arteries, producing enveloped infectious virions. However, unlike the effects of CMV on fully permissive human lung fibroblasts, the effects of strain AD-169 on SMC cultures were delayed and prolonged, resulting in extended survival of a fraction of the starting population. This period of survival did not exceed the life-span of the control SMC cultures. Infectious CMV continued to be isolated from the surviving SMC cultures after extinction of the original inoculum by dilution and after treatment of the cultures with CMV neutralizing antibody. The implications of these findings for the pathogenesis of atherosclerosis are discussed.

Presence of retrovirus in the B95-8 Epstein-Barr virus-producing cell line from different sources.

The B95-8 cell line, a widely used source of highly transforming Epstein-Barr virus (EBV), obtained from the laboratory of origin, harbored an infectious retrovirus. This retrovirus generally resembled the Type D retroviruses structurally and developmentally and like the Type D retroviruses preferred Mg2+ to Mn2+ in its RNA-directed DNA polymerase reaction. Evidence for the presence of retrovirus was found in B95-8 cultures from two other sources within the United States, either by assay for polymerase or by electron microscopy. Comparison of two B95-8 cell lines showed cytogenetic differences as well as differences in retroviral activities. The results suggest that any B95-8 culture should be tested for the presence of retrovirus before its use as a source of EBV.

Natural cell-mediated cytotoxic activity in the peripheral blood of patients with atopic dermatitis.

Ten patients with atopic dermatitis and ten healthy control subjects, matched for age, sex, and skin color, were studied for their peripheral blood natural cell-mediated cytotoxic activity against K-562 human leukemia cell line target cells, using the sodium chromate Cr 51 release assay. There was a great reduction in the natural cytotoxic activity of the patients with atopic dermatitis compared with that of their respective control subjects; the patients with atopic dermatitis had a mean value of 41.9 alpha +/- 28.3% of that of the control subjects.

Genetic susceptibility and resistance to diet-induced atherosclerosis and hyperlipoproteinemia.

To test the hypothesis that genetic susceptibility or resistance to diet-induced atherosclerosis is correlated with serum levels of specific lipids, lipoproteins, or apoproteins, male mice of a genetically susceptible and a genetically resistant strain were fed either a normal or an atherogenic diet. After 20 weeks on a normal diet, neither the resistant nor the susceptible strain mice had atherosclerosis; resistant strain mice had serum cholesterol of 66 +/- 11 while the susceptible strain mice had 90 +/- 1 mg/dl serum cholesterol, and lipoproteins were dominated by a single alpha-migrating HDL. After 20 weeks on an atherogenic diet, resistant strain mice had 185 +/- 55 mg/dl cholesterol, their lipoproteins remained dominated by alpha-migrating HDL, and two of eight mice had mild atherosclerotic lesions; susceptible strain mice had 510 +/- 94 mg/dl cholesterol, multiple alpha- and pre-beta-migrating lipoprotein species, and all 13 had advanced aortic atherosclerosis. The resistant strain mice had an apolipoprotein E/total lipoprotein protein ratio of 0.42 on the normal diet and 0.53 on the atherogenic diet, while the susceptible strain mice had the significantly lower ratios of 0.07 and 0.31, respectively. These data indicate that genetic resistance to diet-induced aortic atherosclerosis in mice is correlated with capacity to prevent large increases in serum cholesterol, to suppress abnormal alpha- and pre-beta-migrating lipoproteins, and to maintain elevated serum apolipoprotein E/total lipoprotein protein ratios. Our data do not preclude the possibility of additional gene control at the level of arterial end organ response.

lack of suppressor cell activity for natural killer cells in infant, aged and a low responder strain of mice.

We have examined the reported role of suppressor cells in the regulation of NK activity in mice with naturally low NK activity (infant and aged (C57 X A)F1 hybrids (CAF1) and low responder strain AKR mice). Possible suppressor activity was assayed by mixing, at a 1 : 1 ratio, spleen cells from low activity mice with spleen effector cells from normally active 8 to 10 wk old CAF1 mice. The lytic activity of the mixture was compared with the activity of effector cells diluted with medium alone or diluted 1 : 1 with "non-suppressor" population which served as a control for nonspecific decreases in lysis. The control or "filler" cells employed were suspensions of adult CAF1 thymus, thymus from adult mice exposed to 2,000 R, and adult CAF1 spleen cells cultured for 24 hours, a procedure that depleted NK activity. In no case was the activity observed in the presumed suppressor-effector mixture significantly lower than that observed in the filler-effector cell mixtures. Thus, in infant (1 to 2 wk) and aged (12 to 18 mo) CAF1 mice and in 8 to 10 wk old AKR mice, we found no evidence for specific cell-mediated suppression of natural cytotoxicity.

Induction of tumor-specific cell-mediated immunity by a noninfectious type-C virus.

Hamsters immunized with either noninfectious hamster type-C virus (D9) or irradiated D9 tumor cells were tested for cell-mediated immune reactivity by the macrophage migration inhibition assay. The migration of peritoneal exudate cells from immunized hamsters was significantly inhibited by either D9 virus or D9 tumor extract, but not by extract of an unrelated CELO virus-induced tumor. The virus and tumor extracts had little or no effect on the migration of peritoneal exudate cells from normal hamsters. Noninfectious D9 virus produces a cell-mediated immune response in the hamster and shares antigenicity with D9 lymphoma, which releases the virus.

Augmentation of natural killer cell activity in spleens of infant, aged, and low responder strain mice by Corynebacterium parvum.

Murine natural killer (NK) cell activity is both age- and strain-dependent. NK activity does not appear in murine spleen cells until three weeks after birth. Activity peaks at approximately 10 weeks, decreasing thereafter with mice over one year old showing significantly reduced levels. Mice showing low or no NK activity because of age (aged and infant mice, respectively) can be stimulated to show significant levels of NK lysis by i.p. injection of formalin killed Corynebacterium parvum (CP). In addition, CP treatment is also capable of increasing NK activity in mice from the normally low responding AKR strain. The NK activity induced or stimulated by CP appears to be like normal NK reactivity in that it is not decreased by removal of T-cells or adherent cells. Thus, in addition to increasing NK activity in normally responsive mice, CP is capable of augmenting NK activity in mice which normally show low or no levels of NK lysis.

Development of natural killer cell activity and genetic resistance to bone marrow transplantation with age: effect of neonatal thymectomy.

The effect of neonatal thymectomy on the development of splenic and bone marrow natural cell-mediated cytotoxicity and on genetic resistance to bone marrow transplantation was examined in mice. Natural cytotoxicity was measured by a 51Cr release assay; the ability to engraft foreign bone marrow was assayed by the spleen colony method. The natural cytolytic response of spleen cells increased progressively from youth to early adulthood, whereas that of the bone marrow declined during the same age period. Neonatal thymectomy significantly elevated the natural killer cell response of young mice only (4 weeks, spleen; 6 weeks, bone marrow). In other experiments, neonatally thymectomized and sham-operated mice were lethally irradiated at 4 or 6 weeks of age and injected with 2.5, 5.0 or 10 million rat marrow cells. Six days later spleen colonies were markedly reduced in both 4- and 6-week-old neonatally thymectomized mice with all rat marrow cell doses tested. Neonatal thymectomy did not alter the percentage of erythroid verus other colonies at either 4 or 6 weeks. In both thymectomized and sham-operated mice the number of colonies increased with increases in marrow cell dose. The data are suggestive of a production and dissemination to the spleen of cels involved in the natural cytotoxic response from the bone marrow.

Amelioration of graft-versus-host disease by pretreatment of allogeneic cells with Fab fragments.

We have previously reported the protective effect of pretreatment of semiallogeneic marrow and spleen cells with Fab fragments of horse anti-mouse thymocyte globulin against otherwise 100% fatal acute graft-versus-host disease (GVHD). We have now examined the effect of this pretreatment in three allogeneic strain combinations of mice, namely, A leads to C57, CBA leads to CAF1, and CBA leads to BALB/c. Acute GVHD mortality at 15 days postirradiation and transplantation of untreated marrow and spleen cells was 100% in all the strain combinations. However, pretreatment of cells with Fab reduced acute GVHD mortality to 6, 13, and 33% for the CBA leads to CAF1, CBA leads to BALB/c, and A leads to C57 strain combinations, respectively. The combined mortality from both acute and secondary GVHD and all other causes at 60 and 200 days postirradiation was 6 and 11%, 20 and 39%, 50 and 66% for each of these strain combinations, respectively. These results provide additional evidence for the effectiveness of Fab in ameliorating acute GVHD and establish its applicability to allogeneic strain combinations.

Comparative effects of different strains of Corynebacterium parvum on natural cell-mediated cytotoxicity.

Because there are conflicting reports regarding the effects of Corynebacterium parvum (CP) on natural killer (NK) cell activity, several different strains of CP were compared. In replicate experiments, age- and sex-matched mice received 0.25-mg i.p. injections of one of four types of CP; formalin-killed strain 6134; heat-killed strain 6134; formalin-killed strain 5888 (actually Corynebacterium granulosum); or formalin-killed CP from the Pasteur Institute. At various days thereafter, two to three mice from each group were sacrificed to determine spleen weight, cellularity, and NK cell activity versus YAC-1 lymphoma cells. The CP from the Pasteur Institute augmented NK cell activity 3 days following injections; however, the activity returned to normal by Day 7 and remained at that level. On the other hand, strain 5888 did not cause as great an increase in lytic activity as did the Pasteur Institute CP at Day 3; but by Day 10 after injection, NK cell levels were above control, and they remained elevated through Day 21. Both the heat-killed and formalin-killed preparations of strain 6134 stimulated NK cell activity initially but resulted in a loss of activity at the later times tested. Experiments done with different doses and routes of injection yielded similar results. Thus, we were able to demonstrate that different types of CP have different effects on NK cell activity in mice and that the general kinetics of these effects were independent of dose or route of administration.

The abrogation of in vivo resistance to parental bone marrow transplantation and of in vitro natural cell-mediated cytotoxicity to the YAC lymphoma by in vivo growth of a transformed thymus-derived cell culture.

A thymus-derived monolayer culture, referred to as E1, from (C57Bl/6 X A)F1 hybrid mice was continuously passaged in vitro for over three years and formed rapidly growing, non-metastasizing tumors when injected s.c. into syngeneic mice. The spleens of tumor-bearing mice were greatly enlarged, but no tumor cells were detected in these spleens. The natural cell-mediated cytotoxic activity of spleen cells of tumor-bearing mice decreased with increasing tumor size. In addition, the expression of genetic resistance to transplantation of C57Bl/6 parental bone marrow cells was decreased in the spleens of irradiated tumor-bearing mice. This correlation between the expressions of natural cell-mediated cytotoxicity and of genetic resistance to bone marrow transplantation is consistent with the hypothesis that both of these responses are mediated by the same population of effector cells.

Augmentation of erythroid burst formation by the addition of thymocytes and other myelo-lymphoid cells.

Bone marrow from barrier-sustained specific pathogen-free (SPF) CBA and C57BL/6 mice gave relatively low numbers of BFU-E colonies in methylcellulose culture, as compared to conventional mice. Addition of thymocytes to the marrow cultures increased the yield of BFU-E colonies more than fourfold in SPF mice but only 1.5-fold in conventional mice. Colony size was also increased. Increased yield of BFU-E colonies was also obtained by co-culture of bone marrow with lymph node cells or with bone marrow or spleen cells from 900R whole-body irradiated mice. The effect appeared to be cellular rather than humoral. It was not reproduced by conditioned medium from thymus or pokeweed mitogen stimulated spleen cells. The helper effect of thymus cells was eliminated or reduced by freezing and thawing, or by 48 hours of incubation after irradiation. Treatment of bone marrow cells in vitro with anti-theta serum and complement did not decrease the number of BFU-E colonies. The putative helper cells appear not to be T cells, were non-adherent to the plastic culture dish, and were cortisone resistant and radioresistant. The low BFU-E colony yield from SPF mouse marrow is presumed to be largely the result of deficiency of these non-T helper cells in SPF bone marrow, rather than of BFU-E progenitor cells.

Genetic resistance to bone marrow transplantation as a leukemia-lymphoma defense mechanism: correlation of "in vivo" lymphoma resistance and "in vitro" NK activity.

(C57 X AKR) F1 hybrid mice, which show genetic resistance to C57 parental bone marrow cells and to AKR lymphoma but not to AKR bone marrow cells in vivo, also show high natural killer cell lysis versus lymphoma cells in vitro. (C3H X AKR) F1 hybrids which show no genetic resistance to parental bone marrow cells or to lymphoma cells in vivo have low levels of NK activity like those of the low responding AKR strain. Thus genetic resistance to bone marrow transplantation and to lymphoma in vivo, correlates with NK lytic activity versus lymphoma cells in vitro, adding to the evidence linking these phenomena.