Murine cytomegalovirus infection: hematological, morphological, and functional study of lymphoid cells.

Mice were studied for 3 to 4 months after murine cytomegalovirus (MCMV) infection. Serial hematological parameters were evaluated. It was found that MCMV infection of mice were accompanied by the appearance of many atypical lymphocytes similar to those seen in association with the hematological features of mononucleosis associated with human CMV infection. Certain functions of splenocytes were studied in infected and uninfected animals during the 4 months after MCMV infection. Three periods were identifiable by the functional response of splenocytes during the course of MCMV infection. The initial phase was characterized by an elevated response of splenocytes to a T cell mitogen and a B cell mitogen (phytohemagglutinin and lipopolysaccharide, respectively). The intermediate phase was marked by productive virus replication in the salivary gland, injury to lymphoid elements, a depressed response of splenocytes to mitogens (phytohemagglutinin and lipopolysaccharide), and decreased humoral splenocytes to phytohemagglutinin stimulation was again increased, and a nonproductive latent infection was established. Study by scanning electron microscopy of splenocytes during the course of infection revealed morphological changes which were correlated with functional alterations.     

Macrophage-induced thymic lymphocyte maturation.

Guinea-pig peritoneal macrophages were found to influence the functional maturation of thymic lymphocytes. Autologous thymic lymphocytes obtained from macrophage co-cultures responded to three different mitogens and were reduced in their ability to reassociate spontaneously with macrophages. Neither of these properties were found in thymic lymphocytes that had not been cultured with macrophages. These functional changes appeared to be specific for macrophages since thymic lymphocytes incubated with skin fibroblasts failed to respond to the test mitogens. Furthermore, they were not the result of either the inactivation, by macrophages, of a putative suppressor thymocyte or a soluble macrophage product. In addition to influencing the functional maturation of thymic lymphocytes, macrophages also appeared to play a direct role in inducing the mitogen response of functionally mature cells.     

Local immune response in experimental pyelonephritis in the rabbit. I. Morphological and functional features of the lymphocytic infiltrate.

The cellular activity of circulating lymphocytes and lymphocytes isolated from the infected kidney of animals with experimental haematogenous pyelonephritis was evaluated. The incorporation of [3H-methyl]thymidine into DNA by lymphocytes was studied with mitogens such as phytohaemagglutinin (PHA), pokeweek mitogen (PWM) and goat anti-rabbit IgG (GARIG). Lymphocytes from infected kidney had a high baseline DNA synthesis compared to circulating lymphocytes from days 5 to 27 of infection. Infected kidney lymphocytes failed to respond to PHA, PWM, or GARIG, whereas circulating lymphocytes did respond to these mitogens. Uropod-bearing lymphocytes, which were shown to be T lymphocytes, were present from days 5 to 77 of infection. B lymphocytes, as determined by surface immunofluorescent technique, were present by day 12, coincident with the onset of local synthesis of antibody. These studies reveal that in pyelonephritis, the cellular response goes through sequential changes and indicate a dynamic interrelationship between T and B lymphocytes at an infected site.     

Alkaline phosphatase activity is expressed only in B lymphocytes committed to proliferation.

Alkaline phosphatase (APase) activity was measured in murine splenic lymphocytes stimulated with the T lymphocyte mitogens phytohemagglutinin (PHA) and Concanavalin A (Con A) and the B lymphocyte mitogens lipopolysaccharide (LPS) and anti-immunoglobulin (anti-Ig). APase activity was found to be enhanced specifically in mitogen-stimulated B lymphocytes, but not in T lymphocytes. This enhancement starts around 8 h after stimulation with a mitogen. With soluble anti-Ig it was observed that the B cells enter G1 phase as assessed by RNA synthesis and blast transformation. However, these cells fail to synthesize DNA and also do not show any increase in APase activity. When the same anti-Ig coupled to Sepharose was used as a stimulator, cells synthesized DNA and also showed significant increase in APase activity. When hydroxyurea was added, the enhancement in APase activity by the mitogen was not diminished although the cells failed to synthesize DNA. These observations indicate that APase activity is enhanced only in activated B cells committed to proliferation.     

Nonspecific lymphocyte responses in F344 and LEW rats: susceptibility to murine respiratory mycoplasmosis and examination of cellular basis for strain differences.

Mycoplasma pulmonis produces a mitogen which may play a role in the pathogenesis of murine respiratory mycoplasmosis in rats. Since LEW rats are more susceptible to this disease than F344 rats are, these two strains were used to examine a possible association between disease severity and the level of nonspecific lymphocyte stimulation by mitogens, including M. pulmonis membrane preparations. F344 and LEW spleen, lung, blood, and lymph node lymphocytes were exposed to various mitogens. LEW lymphocytes gave a significantly higher response to mitogenic stimulation, regardless of their anatomical source. These differences in lymphocyte responsiveness were primarily due to differences within the nonadherent cell population. Significantly higher numbers of W3/25+ (T helper) cells were found in LEW lymphoid populations, whereas no difference was found in MRC OX-8+ (T suppressor/cytotoxic) cells. These data suggest an association between disease severity and host responsiveness to nonspecific stimuli.     

Synergy between human T and B lymphocytes in their response to phythaemagglutinin and pokeweed mitogen.

Human B- and T-lymphocyte preparations were isolated by separating T lymphocytes that formed rosettes with sheep erythrocytes from unrosetted B lymphocytes. Pokeweed mitogen stimulates the proliferation of both B- and T-lymphocyte preparations. In contrast, phytohaemagglutinin stimulates little or no proliferation of purified B lymphocytes although it stimulates the proliferation of T lymphocytes. Lymphoid preparations containing both T and B lymphocytes are more responsive to both mitogens than are either T- or B-lymphocyte preparations. This observation suggested synergy between T and B lymphocytes in the response of unfractionated lymphocytes to mitogens. The basis for this synergy was shown to be the capacity of T lymphocytes to facilitate the proliferation of B lymphocytes cultured with pokeweed mitogen or phytohaemagglutinin. The activity of T lymphocytes is not dependent upon their proliferation or attributable to their release of mitogenic factors. With regard to the clinical evaluation of immune function, our results indicate that the proliferative response of human lymphocytes to phytohaemagglutinin or pokeweed mitogen cannot be directly related to the percentage of T lymphocytes in the lymphoid preparation.     

Nitric oxide synthesis is depressed in Bos indicus cattle infected with Trypanosoma congolense and Trypanosoma vivax and does not mediate T-cell suppression.

Infection with African trypanosomes causes the diseases sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. Suppression of cellular immune responses is a feature of trypanosomiasis in bovine, human, and murine hosts. Some aspects of immunosuppression in the murine model are mediated by nitric oxide (NO) produced by gamma interferon (IFN-gamma)-activated macrophages. We have investigated whether a similar mechanism is responsible for T-cell unresponsiveness in bovine trypanosomiasis. Bovine monocytes and macrophages from uninfected cattle and activated in vitro with IFN-gamma produced NO; however, this response was down-regulated in infected cattle. Similarly, the expression of inducible NO synthase messenger RNA was depressed in macrophages of infected cattle. Proliferation of mononuclear cells of trypanosome-infected cattle cultured with mitogen or trypanosome antigens was unchanged by the addition of an NO synthase inhibitor. Lymphocytes of infected cattle secreted interleukins with T-cell growth factor activity after in vitro activation with mitogens but not after activation with trypanosome antigens. Although lymph node cells secreted IFN-gamma after in vitro activation, ex vivo expression of mRNA was depressed. In contrast, the level of expression of interleukin 10 mRNA was higher during infection. We conclude that NO is not involved in the loss of T-cell proliferative function associated with trypanosomiasis in cattle and that, in contrast to the mouse model, the capacity of monocytes and macrophages to produce NO is actually down-regulated in infected cattle.     

In vitro and in vivo effects of interferon on the response of human lymphocytes to mitogens

Previous studies have shown that addition of IFN to the assay in vitro inhibits the proliferative response of lymphocytes to mitogens, whereas long term treatment by IFN in vivo has no major effect on the mitogen responsiveness of tumour patient's lymphocytes. Possible reasons for the discrepancy between the results obtained following treatment by IFN in vitro and in vivo were investigated. It was observed that the proliferative response of tumour patients lymphocytes to various mitogens was not affected to any major extent 24 hr after a single injection of 3 million units of interferon-alpha (IFN-alpha). Lymphocytes from tumour patients and healthy donors were found not to differ in their susceptibility to IFNs' anti-proliferative effect in vitro. Pure IFN-beta, present in the assay throughout the incubation period, inhibited the response of lymphocytes to polyclonal mitogens and PPD showing IFN and not contaminants in the preparations to be responsible for this effect. Although the presence of IFN in the assay throughout the incubation period inhibited the proliferative response of lymphocytes, pre-treatment of these cells with IFN-alpha in vitro was found to have no major effect on their response to mitogens. We conclude that the lack of effect on the proliferative response of lymphocytes following treatment by IFN in vivo, is probably due to the fact that the lymphocytes were only treated with IFN prior to the assay.     

Cooperative pathway induction of T lymphocyte mitogen stimulation.

Isolated peritoneal mouse macrophages pretreated with the mitogenic enzyme combination neuraminidase (EC 3.2.1.18) plus galactose oxidase (EC 1.1.3.9.) (NAGO), or with NaIO4, stimulate macrophage-depleted lymphocytes mitogenically by a macrophage-derived signal, different from the originally used mitogen. Polyethylene glycol (PEG) treatment of the cultures, although itself nonmitogenic, strongly enhances the mitogenic response of the lymphocytes. Under culture conditions the macrophage-derived signal is transmitted to lymphocytes by direct cell contact, a finding which explains the need of a critical cell density for T lymphocyte stimulation. In the absence of macrophages, lysates from mitogen-preteated macrophages stimulate column-purified lymphocytes in the presence of PEG. Our results indicate that mitogenic activation of lymphocytes is mediated through two sequential triggering events, induction (by mitogen treatment) of a macrophage-derived signal and commitment (by nonmitogenic PEG treatment) of lymphocytes to react to the signal. Reconstitution of the mitogenic response can be achieved by a sequential induction of both these events.     

Age-related changes in ConA- and LPS-induced lymphocyte transformation. I. Effect of culture conditions on mitogen responses of blood and spleen lymphocytes from young and aged rats.

Age-related changes in LPS- and ConA-responsiveness of rat spleen lymphocytes as judged by 14C-TdR incorporation were studied. It was found that responses to both mitogens decreased with advancing age. This report shows that the reduced 14C-TdR incorporation could not be attributed to decreased cell survival or viability of spleen lymphocytes from old rats, to delayed proliferation of the old lymphocytes, or to differences in minimum mitogen doses required for optimal stimulation. The results suggest that the observed decrease is due to a decrease in the number of mitogen responsive cells. The response to LPS was even more depressed than was the response to ConA. The response to ConA in whole blood is also shown to decline with ageing at multiple mitogen doses.     

Effects of irradiation upon the response of murine spleen cells to mitogens.

The mitogenic response of murine spleen cells exposed to graded doses of radiation was evaluated. Low-dose exposures were associated with an augmented response to concanavalin A (Con A) that was most marked with 100 rads. Low-dose augmentation of phytohemagglutinin (PHA) stimulation was equivocal and most pronounced in cells exposed to 10-20 rads. Augmentation was only demonstrable when the cells were irradiated immediately prior to mitogenic stimulation. Timed exposures after stimulation with Con A or PHA showed no evidence that mitogen activation increased radioresistance, although the possibility could not be excluded that activation protects against interphase cell death. Reduced isotope incorporation was associated with all doses of radiation evaluated in cells stimulated with lipopolysaccharide (LPS) or pokeweed mitogen (PWM). On this basis it is concluded that 1) each of the mitogens tested differs in its capacity to stimulate irradiated spleen cells; 2) radiation-induced augmentation is noted with those mitogens (Con A and possibly PHA) known to activate only T cells; 3) radiation-induced augmentation may be due to the release of mitogenically active molecules by injured lymphocytes.     

Changes in the phenotype of T-cell subset determinants following murine cytomegalovirus infection

The murine model provides a particularly apt experimental system in which to evaluate the effects of cytomegalovirus (CMV) infection. CMV exerts a profound "suppression" of the immune response in the mouse and in humans; the infected animal is no longer able to mediate an appropriate response to mitogens or alloantigens. Using fluorocytometry and fluoresceinated monoclonal antibodies directed against the Thy-1.2, Lyt-1, and Lyt-2 cell membrane determinants following a nonlethal intraperitoneal inoculation of weanling BALB/c mice with Smith strain murine cytomegalovirus, significant changes in T-cell subsets were found that are consistent with findings described in the clinical situation. These ratio changes are temporally consistent with the cytotoxic T-lymphocyte population described by others. Finally, novel changes in the antigenic determinant distribution is found which may reflect the appearance of an antigen-committed cytotoxic T-lymphocyte population. This population which peaks at the ninth postinfection day may consist of 20-47% of the T-lymphocyte population and may offer an explanation for the cellular hyporesponsiveness seen following CMV infection.     

Kinetics of inhibition of mitogen-induced proliferation of human lymphocytes by alpha 2-macroglobulin in serum-free medium

The effect of alpha 2-macroglobulin (alpha 2M) on the ability of human lymphocytes to proliferate in response to stimuli by 3 mitogens, concanavalin A, phytohaemagglutinin and pokeweed mitogen was investigated in in vitro lymphocyte cultures in serum-free medium. The following experiments were performed: 1. lymphocytes were treated with alpha 2M prior to stimulation with the mitogens; 2. alpha 2M and mitogens were added simultaneously to the lymphocyte cultures; and 3. alpha 2M was added to the lymphocyte cultures after they were stimulated with the mitogens. In all cases, alpha 2M was found to inhibit mitogen induced proliferation of the lymphocytes as evaluated by (6-3H)-thymidine uptake of the cells. The mechanisms involved in the inhibition of lymphocyte proliferation by alpha 2M are discussed.     

The reactivity of spleen cells from malarious rats to non-specific mitogens.

The reaction of spleen cells from rats infected with Plasmodium berghei to non-specific mitogens has been measured. The cells have been stimulated in vitro by phytohaemagglutinin, concanavalin-A and by bacterial lypopolysaccharide. In addition the release of lymphocyte activating factor (LAF) by splenic macrophages has been assayed using a heterologous thymocyte culture. The reactivity of spleen lymphocytes from malarious rats is severly affected. The cells do not react either to the T cell-specific mitogens or to the B-cell stimulant. The reactivity of macrophages, as measured by the release of LAF, was not altered by the disease.     

Changes in the population of lymphocytes and their response to mitogens during the growth of a Simian virus 40-induced fibrosarcoma in hamsters.

Changes have been studied occurring in spleen and thymus lymphocytes, as well in their response to different mitogens, during the growth of Simian virus 40-induced fibrosarcomas in hamsters. During tumor growth, the spleens of tumor-bearing animals became considerably enlarged, and their content in lymphoid cells increased by 100%. Spleen lymphocytes responded normally or slightly higher than did normal controls to concanavalin A (Con A) stimulation when tumors were small, but their response gradually decreased to 30–40% of normal in animals bearing large tumors. These same cells, when cultured with normal lymphocytes in the presence of Con A, were without effect, or were mildly suppressive of the normal cellular response to Con A. The response to Iipopoly-saccharide (LPS) decreased slightly. The thymus, on the other hand, became smaller and depleted of cells, and here too, the response to Con A gradually diminished. Macrophages were found in increased numbers in the enlarged spleens of tumor-bearing animals. It is suggested that they played a role in the suppression of lymphocyte response to mitogens. Elimination of macrophages from cultures by the addition of carageenan resulted in an enhanced response of lymphocytes from tumor-bearing animals to Con A. In addition, we found that carrageenan acts as a mitogen, most probably for B cells, and that when added to cultures containing LPS, a potentiation of the mitogenic response to LPS was obtained.     

Suppression of lymphocyte proliferation by parainfluenza virus type 3-infected bovine alveolar macrophages.

Lymphocytes stimulated with concanavalin A (Con A) or antigen in the presence of bovine parainfluenza virus type 3 (PIV-3) infected bovine alveolar macrophages (BAM) or monocytes, had depressed [3H]thymidine incorporation. This failure of lymphocytes to incorporate radiolabel required live virus, was time dependent and was most pronounced when BAM were infected for 48 hr prior to the addition of lymphocytes. The rate of infection of alveolar macrophages and the release of infectious virus into culture supernatants paralleled suppression of lymphocyte mitogenesis by PIV-3. However, the peak titre of exogenous, live or inactivated virus was not suppressive when added to lymphocyte macrophage cultures just prior to Con A stimulation. Neither the loss of viable alveolar macrophages nor a shift in antigen or mitogen dose response in virally infected cultures could account for the deficit in [3H]thymidine incorporation by lymphocytes. Despite the presence of lymphocyte-associated virus antigen detected by direct immunofluorescence, no increase in PIV-3 titre above baseline was seen from infected lymphocytes, irrespective of mitogen stimulation. Likewise, lymphocytes did not contribute to the extracellular virus pool in lymphocyte-macrophage cultures as the increases in viral titre above basal levels in supernatants were equal to levels released by macrophages alone. The expression of viral antigen on lymphocytes stimulated in the presence of PIV-3-infected BAM suggests a non-productive or abortive infection of lymphocytes mediated through contact with infected macrophages.     

Development of a serum-free medium for in vitro immune responses by using beta-cyclodextrin. Demonstration of the requirements for polyamines

A serum-free medium has been developed which is able to support primary antibody responses by cultured murine lymphocytes. This medium is based on RPMI 1640 that is supplemented with beta-cyclodextrin, insulin, transferrin, albumin, low density lipoprotein, putrescine and L-alanine as substitutes for fetal calf serum. Omission of anyone of these components resulted in a marked decrease of antibody responses. By employing the serum-free culture conditions, it was clearly demonstrated that polyamines such as putrescine, spermidine and spermine had positive effects on the development of antibody-forming cells. This serum-free medium supported the antibody response to sheep erythrocytes, trinitrophenyl (TNP)-Ficoll or TNP-lipopolysaccharide as efficiently as 10% fetal calf serum-containing medium. In addition, murine lymphocytes proliferated in response to an antigen or a mitogen equally well in these two types of medium.     

In vitro immunomodulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the cellular immune response

The aim of this study was to determine the immunomodulatory activity of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide in vitro. This compound was used for the synthesis of a series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides with documented immunotropic activity. The performed measurements assessed the cytotoxic effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the murine macrophages (cell line J774E.1) and lymphoblasts (cell line D10.G4.1), the influence of this compound on the proliferation of murine lymphocytes isolated from peripheral lymphatic organs and murine peritoneal macrophages stimulated with mitogens (concanavalin A(ConA), lipopolysaccharide (LPS), phytohemagglutinin A (PHA)). Moreover, the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β by the murine peritoneal macrophages stimulated with LPS from Escherichia coli was assessed. It was found that 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide displayed no cytotoxic effects in the murine J774E.1 and D10.G4.1 cell lines in a wide range of concentrations (0.5–200?μg/ml). Furthermore, the compound stimulated proliferation of lymphocytes isolated from the spleen and mesenteric lymph nodes when used alone and in combination with mitogens (ConA and PHA). This effect was stronger in the nonstimulated cells, and it followed a dose–response relationship. The same phenomenon was observed for the proliferation of the murine peritoneal macrophages. The investigated hydrazide, at the highest used concentration of 150?μg/ml, increased the LPS-induced production of IL-1β and did not affect the level of TNF-α. These results confirmed the immunomodulatory properties of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide and indicated that this compound could be useful in further research aimed at finding novel functional drugs.     

Replication of murine cytomegalovirus in lung macrophages: effect of phagocytosis of bacteria.

Murine cytomegalovirus was found to replicate in lung and peritoneal macrophages of both CF-1 and BALB/c mice in vitro. Cytopathic changes typical of cytomegalovirus infection, including intranuclear inclusions, developed within the infected cells and eventually resulted in death of infected macrophages. Viral antigens were demonstrable by indirect immunofluorescence microscopy, and morphologically typical herpesvirus particles were observed in both nuclei and cytoplasm of murine cytomegalovirus-infected macrophages. Within 24 h after infection, at which time there was expression of viral antigens but no marcophage death, murine cytomegalovirus-infected macrophages demonstrated marked inhibition of phagocytosis of Staphylococcus aureus. Direct inhibition of macrophage function by cytomegalovirus infection in vivo could impair pulmonary defenses and may account in part for the frequent association of cytomegalovirus infection with other infectious agents.     

Selective response of lymphocytes from Treponema pallidum-infected rabbits to mitogens and Treponema reiteri.

The in vitro response of peripheral blood lymphocytes from rabbit infected with Treponema pallidum was examined using various mitogens and avirulent Treponema reiteri. For the first 4 weeks after treponemal infection, the response of lymphocytes from syphilitic rabbits to phytohemagglutinin and pokeweed mitogen was markedly reduced in comparison to uninfected controls. Lymphocytes from both groups of rabbits responded normally to class-specific immunoglobulin anti-sera (anti-immunoglobulin M and anti-immunoglobulin G) and T. reiteri.