Flow cytometric analyses of the specific activation of peripheral blood mononuclear cells from healthy donors after in vitro stimulation with a fermented mistletoe extract and mistletoe lectins

Immunostimulatory properties of mistletoe extracts derived from Viscum album L. (VAL) are well described, demonstrating activation especially of T, T-helper cells and monocytes/macrophages. In order to characterise in detail the communication between different cell populations, we studied mistletoe-induced expression of co-stimulatory signals and their ligands by flow cytometry. Peripheral blood mononuclear cells (PBMC) from 15 healthy controls were incubated for 7 days with a fermented VAL extract. VAL significantly upregulated the expression of the co-stimulatory molecule B7.1 (CD80) on monocytes/macrophages, but not B7.2 (CD86). No significant changes in the expression of either molecules on B cells could be found, suggesting that only monocytes/macrophages act as antigen presenting cells (APCs) in this in vitro system. Purified mistletoe lectins, components of most VAL extracts were also analysed, but did not induce similar responses of monocytes/macrophages. The receptor for B7 molecules, CD28, but not CTLA-4 (CD152), was also found to be significantly enhanced on CD4+ cells after VAL simulation. There was no evidence for activation of a B cell response via the CD40/CD40L pathway. Our data support the concept that stimulation by VAL extracts induces a specific T-helper cell reaction with monocytes/macrophages acting as APCs and purified lectins do not exert the same effects.     

Lymphoproliferative immune function in the Sydney Blood Bank Cohort, infected with natural nef/long terminal repeat mutants, and in other long-term survivors of transfusion-acquired HIV-1 infection

OBJECTIVES: To assess T-helper cell immune function (proliferation) in members of the Sydney Blood Bank Cohort (SBBC) compared with other individuals with transfusion- and sexually acquired HIV-1 infection and with matched HIV-negative controls. DESIGN AND METHODS: Decreasing CD4 counts and T-helper cell function are associated with disease progression. Peripheral blood mononuclear cells (PBMC) from study subjects were assayed for in vitro proliferative responses to HIV-1-derived antigens, recall antigens and alloantigen. T-helper cell function and CD4 counts in members of the SBBC were followed longitudinally. RESULTS: Proliferative responses and CD4 counts from members of the SBBC were similar to or better than those of other transfusion- or sexually-acquired HIV-1-positive long-term non-progressors (LTNP), including the HIV-negative matched SBBC control groups. However, individuals with disease progression had reduced or undetectable proliferative responses to recall antigens but a conserved response to alloantigen; they also had low CD4 counts and low CD4:CD8 ratios. In the SBBC, these immune parameters were usually stable over time. CONCLUSIONS: The unique SBBC with natural nef/long terminal repeat deletions in the HIV-1 genome were genuine LTNP without showing signs of disease progression. They appeared to be a group distinct from the tail-end of the normal distribution of disease progression rates, and may remain asymptomatic indefinitely. The SBBC virus may form the basis of a live attenuated immunotherapeutic or immunoprophylactic HIV vaccine.     

Target-cell specificity of fusogenic liposomes: membrane fusion-mediated macromolecule delivery into human blood mononuclear cells

Fusogenic liposome, a unique vector prepared by fusing ultraviolet-inactivated Sendai virus and liposome, is known to efficiently deliver content into various animal cells through membrane fusion. In this study, we examined the target-cell specificity of fusogenic liposome (FL)-mediated macromolecule delivery into human blood cells using diphtheria toxin fragment A (DTA) as a probe. Among the peripheral blood mononuclear cells (PBMC), FL was able to deliver its encapsulates into CD14+ monocytes and CD4-/CD8- T-cells, but not into CD19+ B-lymphocytes, CD4+ T-cells or CD8+ T-cells. The susceptibility of human leukemia cell lines to FL was similar to that of PBMC; the order of the reactivity was U937 (monoblastic leukemia)>MOLT4, Jurkat (T-lymphoma)>Daudi, BALL1 (B-lymphoma)>K562 (erythroblastic leukemia). Interestingly, FL showed similar binding activity to all of these leukemia cell lines. These findings indicate that, among blood cells, monocytes, monoblastic leukemia cells, CD4-/CD8- T-cells and T-lymphoma cells are preferable targets for FL-mediated macromolecule delivery. This is the first demonstration of the existence of non-permissive cells against FL. Our results also suggest that some molecules on target-cells other than the binding targets of SV-derived protein may participate in fusion between FL and cells.     

Lymphocyte subsets and mitogen stimulation of blood lymphocytes in normal pregnancy

PROBLEM: In normal pregnancy the maternal immune system should be directed towards tolerance or suppression in order not to reject the partly foreign feto-placental unit. The aim of this investigation was to find hallmarks of systemic immunosuppression during normal pregnancy. METHODS: Five healthy primigravidae were examined during pregnancy and postpartum with flow cytometric analysis to define T and B lymphocyte subsets in peripheral blood. In addition, we studied the proliferative response of lymphocytes to mitogens or interleukin-2 (IL-2) alone or in combination with immunomodulating drugs or interleukin-4 (IL-4). The results were compared to healthy, non-pregnant women. RESULTS: During pregnancy and early puerperium we noted an immune balance in favour of suppression, as measured by increased numbers of T "helper/suppressor" (CD4+CD45RA+) and "suppressor"/effector T cells (CD8+S6F1-), and decreased numbers of T "helper/inducer" (CD4+CD29+), T "helper/memory" (CD4+CD45RO+), killer/effector T cells (CD8+S6F1+), and Natural Killer cells (CD56+), as well as decreased numbers of activated lymphocytes expressing IL-2 receptor (CD25+) and T cells expressing HLA-DR (HLA-DR+CD3+). During pregnancy, lymphocyte proliferation was impaired in autologous serum with concanavalin A (ConA), phytohemagglutinin (PHA), or IL-2. A difference in proliferative response to PHA or IL-2 between cultures with AB serum and autologous serum is suggestive of an immunosuppressor factor in serum during pregnancy. Indomethacin significantly increased lymphocyte proliferation in autologous serum with ConA, indicating PGE2 mediated suppressor activity during pregnancy. Chlorambucil and cimetidine modulated the proliferative response to ConA, indicating an alkylating agent sensitive and a histamine dependent suppressor activity during pregnancy. CONCLUSIONS: During normal pregnancy, a state of systemic suppression of the maternal immune system seems to be present.     

Alterations of immune functions in heroin addicts and heroin withdrawal subjects

Conflicting results, both decreased and increased, have been reported concerning the function of T-lymphocytes in heroin addicts. We investigated the alterations of T-lymphocyte proliferative responses and immunophenotypic markers on lymphoid cells in heroin addicts and during different periods of heroin withdrawal in addicted subjects. This study has demonstrated a decrease in the response of T-lymphocytes to 1.2, 2.5, 5 and 10 microg/ml of phytohemagglutinin stimuli in heroin addicts and 1- to 5-day heroin withdrawal subjects compared with controls. Similarly, in an in vitro study, 10(-4), 10(-6) and 10(-8) M concentrations of morphine were shown to suppress 0.6 and 2.5 microg/ml of PHA-stimulated T-lymphocyte obtained from naive subjects. This inhibitory effect of morphine on PHA stimulation was completely abolished by 100 microM naloxone. The immunological parameters of total T-lymphocytes (CD3), T-helper cells (CD4), cytotoxic T-cells (CD8), B-cells and natural killer cells that are the immunophenotypic markers studied by flow cytometric analysis were altered in heroin addicts, 15- to 21-day and 6- to 24-month heroin withdrawal subjects, when compared with controls. These results suggest that heroin addicts and short period (15 to 21 days and 6 to 24 months) of heroin withdrawal have decreases in their immune system functioning and that the heroin withdrawal subjects seem to gradually reverse their immunological parameters to normal levels when withdrawal was sustained >/=2 years. This is the first report examining immune function in heroin withdrawal subjects using the "cold turkey" method. The results are beneficial for further study of the mechanism responsible for the opioid-induced changes in immune function.     

Alpha beta T cell response to Toxoplasma gondii in previously unexposed individuals

The mechanisms by which T cells from previously unexposed hosts respond in vitro to certain intracellular pathogens remain to be fully understood. We report and characterize the in vitro reactivity to Toxoplasma gondii of human alpha beta T cells from T. gondii-seronegative individuals. Resting alpha beta T cells from these individuals proliferated in response to PBMC infected with T. gondii or pulsed with T. gondii lysate Ags. This was accompanied by an increase in the percentage of CD4+ alpha beta T cells. Purified CD4+ alpha beta T cells but not CD8+ alpha beta T cells proliferated in response to these T. gondii preparations. Both CD4+ alpha beta T cells with naive (CD45RA+) and memory (CD45RO+) phenotypes from adults as well as alpha beta T cells from T. gondii-seronegative newborns proliferated after incubation with T. gondii. This alpha beta T cell response to the parasite was inhibited by anti-HLA-DR mAb and to a lesser degree by anti-HLA-DQ mAb. Use of paraformaldehyde-fixed PBMC completely abrogated the proliferation of alpha beta T cells, indicating the need for processing of T. gondii Ags. Analysis of the TCR V beta expression did not show evidence for restriction in TCR V beta usage during T. gondii stimulation of alpha beta T cells. Alpha beta T cells secreted significant amounts of IFN-gamma after incubation with T. gondii-infected monocytes. This rapid and remarkable alpha beta T cell response may play an important role in the early events of the immune response to T. gondii.     

Lymphocyte activation by liposome-trapped streptozotocin in murine popliteal lymph node (PLN) test

The lymphoproliferative potential of liposome-trapped streptoztocin (STZ) was compared to the effect of saline-dissolved STZ injected locally into the foot pad of CD-1 mice. Popliteal lymph node (PLN) enlargement and early cell activation of lymphocyte subsets were monitored during the onset of STZ-induced autoimmune-like reaction. Injection of the optimal STZ dose, 0.5 mg/foot pad, markedly increased the absolute PLN cell number as well as specific T-helper (CD4+), T-suppressor/cytotoxic (CD8+), and B-(Ig+) cell subsets stained with fluorescent monoclonal antibodies. Furthermore, there was a marked increase in the number of large/activated CD4+ and CD8+ cells and subsets bearing specific markers of early activation. These included cells stained with fluorescein-conjugated monoclonal antibodies against interleukin-2 receptor (CD25+) and early activation marker (EAM+) (CD69+), and with fluorescein-conjugated peanut agglutinin (PNA+). Surprisingly, the injection of liposome-trapped STZ, at a 1/10 of the optimal dose only, induced a marked PLN enlargement comparable to the effect of optimal STZ dose. The effect of liposome-STZ could be dissociated from the non-drug-containing MLV-related lymphocyte activation. The data suggest several possible advantages from the introduction of chemicals by the liposome route and the subsequent PLN test for chemical-induced autoimmunity. Toxicological advantages could involve better control of chemical exposure, controlled exposure to the water-insoluble substances, drastic reduction of xenobiotic dose, a stronger, clear PLN response and possible elimination or at least restriction of false-negative results, due to the liposome adjuvancity. Overall, application of liposomes as an exposure route potentialized the STZ-induced early lymphocyte activation.     

Band shift analysis of three base-pair repeat alleles in the short tandem repeat locus D12S391

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.     

The importance of CD54 and CD86 costimulation in T cells stimulated with Candida albicans and Dermatophagoides farinae antigens in patients with atopic dermatitis

A number of studies have demonstrated an increased frequency of allergen-specific T cells producing increased amounts of interleukin-4 (IL-4) and IL-5, but little interferon-y in both the peripheral blood and skin lesions of patients with atopic dermatitis (AD). In this study, to further clarify the characteristics of T cells obtained from AD patients, we examined the dependency of the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) from AD patients on costimulatory molecules. The antigens used were Candida albicans and Dermatophagoides farinae, for which AD patients show increased levels of IgE antibodies. PBMC from control healthy donors stimulated with these antigens incorporated [3H]-thymidine much more than PBMC from AD patients. The addition of anti-CD54, -CD40, -CD80 and -CD86 monoclonal antibodies to the cultures showed that the PBMC required only CD54 and CD86 for stimulation with C. albicans, but required CD54, CD80 and CD86 for stimulation with D. farinae. Among these monoclonal antibodies, the anti-CD54 antibody suppressed the proliferative responses of most PBMC, most effectively followed by the anti-CD86 antibody. However, there were no significant differences in the requirement for costimulatory molecules of PBMC proliferation stimulated with C. albicans or D. farinae between AD patients and healthy donors. Since many studies have suggested that T-helper type 1 and T-helper type 2 immune responses are different in their dependency on CD80 or CD86 costimulation, our present results suggest that the allergen-specific T cells of AD patients are not completely shifted to a T-helper type 2 subset.     

Effects of sleep and circadian rhythm on human circulating immune cells

The role of nocturnal sleep for normal immune regulation and its relation to circadian rhythm was examined in 10 men participating in two 51-h sessions. One session included two regular wake-sleep cycles; the other included a night of sustained wakefulness followed by a night of recovery sleep. Blood was collected every 3 h to determine PBMC counts, including the enumeration of monocytes, NK cells, and lymphocyte subsets (CD19+, CD3+, CD4+, CD8+, HLA-DR+). Production of IL-1beta, TNF-alpha, IL-2, and IFN-gamma was determined after stimulation of whole blood samples with LPS and PHA, respectively. Concentrations of IL-6 and cortisol were assessed in plasma. Enumeration of cells indicated significant circadian rhythms for all PBMC subsets under conditions of sustained wakefulness. Compared with sustained wakefulness, nocturnal sleep acutely reduced the numbers of monocytes, NK cells, and counts of all lymphocyte subsets. However, in the afternoon and evening of the day following sleep, counts of NK cells and lymphocytes were significantly higher than after nocturnal wakefulness, indicating that effects of sleep interacted with those of the circadian pacemaker. Sleep markedly enhanced production of IL-2 by T cells (CD3+) but did not influence production of IL-1beta and TNF-alpha, or IL-6 concentrations. Effects of sleep were not mediated by changes in cortisol. The decrease in monocytes, NK cells, and lymphocytes, together with an increased production of IL-2 during sleep, may serve to support ongoing immune defense in extravascular lymphoid tissue during a time of diminished acute Ag challenge.     

In vitro prevention and reversal of lipopolysaccharide desensitization by IFN-gamma, IL-12, and granulocyte-macrophage colony-stimulating factor

LPS tolerance is characterized by a diminished monocytic synthesis of TNF-alpha and, interestingly, IL-10 after LPS restimulation. We wondered whether granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, and IFN-gamma can prevent or reverse this down-regulation of TNF-alpha and IL-10 production. The LPS-induced TNF-alpha amounts in desensitized PBMC treated with GM-CSF, IFN-gamma, or IL-12 and in naive, non-cytokine-primed cultures were similar, while much more TNF-alpha was induced in cytokine-primed naive cells. The effect of IL-12 was dependent on the presence of nonmonocytic cells and could be completely blocked with an IFN-gamma antiserum. Treatment of LPS-desensitized pure monocytes with IFN-gamma or GM-CSF resulted in a very high TNF-alpha expression and no difference to cytokine-primed naive monocytes was evident any longer. While IFN-gamma and IL-12 decreased IL-10 expression in naive PBMC, it was increased by both and by GM-CSF in LPS-tolerant cultures. Again, only IL-12 was dependent on the presence of nonmonocytic cells. For prevention of LPS tolerance, similar results were obtained. Recently, we have shown that IL-10 and TGF-beta mediate LPS desensitization in vitro and can be used to establish LPS hyporesponsiveness in the absence of LPS. IFN-gamma and GM-CSF prevented and reversed down-regulation of TNF-alpha and IL-10 synthesis also in the model of IL-10/TGF-beta1-induced LPS hyporesponsiveness, while IL-12 was ineffective because of its obvious inability to induce IFN-gamma. In summary, after LPS desensitization/hyporesponsiveness, IFN-gamma and GM-CSF tended to normalize pro- and anti-inflammatory monocytic behavior. Our results suggest that during LPS desensitization/hyporesponsiveness, monocytes acquire a hitherto unknown functional state with an altered reaction to biologic response modifiers.     

In situ characterization of leucocyte subpopulations after infection with Eimeria tenella in chickens

We characterized the leucocyte subpopulations after infection with Eimeria tenella in both naive and immune chickens. Immunocytochemical staining was used to characterize the cells in situ, so that the interaction between host and parasite could be studied. More leucocytes were detected in the lamina propria of immune chickens, and leucocytes infiltrated the ceca more rapidly than in naive chickens, but the infiltration was less pronounced than in naive chickens. In naive chickens, most infiltrated leucocytes were macrophages and T cells. Two days after inoculation the number of CD4+ cells had increased greatly. In immune chickens, mainly T cells (CD4+ and CD8+) infiltrated the lamina propria, and in contrast to naive chickens, the number of CD8+ cells exceeded the number of CD4+ cells. Furthermore, we characterized which cells contained a parasite and which cells were detected next to the parasites, because these cells are probably involved in the arrested development of the parasites. In naive chickens, sporozoites were significantly more often located within or next to macrophages than in immune chickens. In immune chickens, sporozoites were significantly more often located within or next to CD3+, CD8+, and TCR2+ cells. In conclusion, the marked increase of CD4+ cells after primary infection suggests that these cells are involved in the induction of the immune response, whereas the increase of CD8+ cells after challenge infection suggests that these cells act as effector cells.     

Primary surgical repair of traumatic lacerations of the lacrimal canaliculi

Normal human peritoneal cells collected during elective laparatomy from patients with gallbladder stones without clinically detectable inflammatory changes were characterized phenotypically with immunocytochemical method and flow cytometry, with special attention paid to the presence of memory cells. The responsiveness of normal PCs to mitogen and, specifically, the role of peritoneal macrophages in this process was studied. The peritoneal cells consisted of 45% of monocytes/ macrophages (CD68+), as many as CD2+ T lymphocytes, 8% CD57+ NK and K 2% CD22+ B, cells. The CD4/CD8 ratio was 0.4. The peritoneal cells did not express interleukin-2 (CD25+) and transferrin receptors (CD71+) on their surface. Approximately 49% of the peritoneal cells were class II MHC antigen positive cells. Two per cent of S100+ dendritic cells were found. Flow cytometric two-colour analysis revealed that the majority of peritoneal CD4+ (92.4%) and CD8+ (73.1%) lymphocytes, while only 50.2% of CD4+ and 30.1% CD8+ peripheral blood cells expressed simultaneously the CD45R0 (UCHL1) molecule, which is characteristic to the memory/effector T-cell subpopulation. Peritoneal T lymphocytes responded to the mitogens less than peripheral blood lymphocytes of the same individual. Supplementation of cell culture with anti-macrophage (anti-CD68) and anti-HLA-DR MoAb brought about a dose-dependent decrease of proliferative peritoneal cell response to ConA. The authors conclude that human peritoneal cell population comprises a high proportion of T lymphocytes and macrophages capable of presenting antigens to peritoneal lymphocytes. High prevalence of memory lymphocytes points to the preparedness of these cells to react with invading antigens most likely of gut bacterial origin.     

Effect of a mistletoe extract (Iscador QuFrF) on viability and migratory behavior of human peripheral CD4+ and CD8+ T lymphocytes in three-dimensional collagen lattices

Migration and tissue distribution of immunocompetent cells may be critical prerequisites for efficient immune surveillance. The effect of various concentrations of the mistletoe extract Iscador QuFrF on the locomotory behavior and viability of immunomagnetically isolated human CD4+ and CD8+ T lymphocytes within three-dimensional collagen gels was investigated. Although variation in baseline activities of spontaneously migrating T cells was donor-dependent, a dose-dependent stimulation of the locomotory activity in both CD4+ and CD8+ T cells for noncytotoxic concentrations of Iscador QuFrF (0.25-1.25 micrograms/ml) was detected. The optimal concentration of mistletoe extract and time of maximal response were specific for each donor. As shown by cell tracking and subsequent data analysis, CD4+ T cells exposed to the mistletoe extract displayed a significant increase in mean velocity and time locomoting; total distance migrated was nearly doubled. In contrast, CD8+ T cells showed less pronounced changes in these critical parameters. Cytotoxic effects of the mistletoe preparation on T lymphocytes, which could at least partially be attributed to the induction of apoptosis, were drastically reduced in the presence of fetal calf serum in the culture system. Our data suggest that the direct stimulation of T-cell migration in the presence of mistletoe components may modulate in a dose-dependent manner the system of immune surveillance and recognition in patients under mistletoe therapy.     

Immunoprotective activity of the galactoside-specific mistletoe lectin in cortisone-treated BALB/c-mice

Hydrocortisone-acetate (HA)-treatment of BALB/c-mice induced a profound suppression of the lymphatic immune system with statistically significant decreases of thymocyte proliferation and maturation rates as well as peripheral blood lymphocyte (PBL) counts. To check its putative immunoprotective/immunorestoring activity, the optimal immunomodulating dosage of commercially available mistletoe extract standardized for the galactoside-specific mistletoe lectin (ML-1) was regularly administered (1 ng ML-1 per kg body weight; subcutaneously). As compared to counts of thymic lymphatic subsets of HA-treated mice, a considerable up regulation of mature cells expressing helper/inducer (L3T4+) as well as cytotoxic/suppressor (Lyt-2+) phenotypes and immature cells expressing both antigens (L3T4+/Lyt-2+) could be found after ML-1 co-administration. Counts of BALB/c-mouse peripheral blood mononuclear cells also revealed statistically significant increases after ML-1 co-administration, as compared to HA-treated animals. Accordingly, ML-1 treatment may be supposed to restore the lymphatic system after immunosuppressive corticoid treatment and thus this treatment may be of great benefit to patients.     

Immunity to porcine rubulavirus infection in adult swine

The immune response against the porcine rubulavirus was analyzed in experimentally infected adult pigs. High titers of virus neutralizing and hemagglutinating inhibitory antibodies were identified in infected animals. The antibody specificity was directed towards HN, M, and NP rubula virion proteins; immunodominance of HN proteins was demonstrated. Peripheral blood mononuclear cells from infected, but not from non-infected pigs proliferated in vitro in response to virus antigenic stimuli, showing a bell-shaped plot with the highest peak at 5 weeks post-infection. Virus-induced lymphoblasts expressed CD4+ CD8+ phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4+ CD8- cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1+) and total T lymphocytes (CD2+) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4+ CD8- blood cells was observed at 3 weeks post-infection, whereas both CD4- CD8+ and CD4+ CD8+ cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4+ CD8+ T cells in the control of porcine rubulavirus infection.     

Both activated and nonactivated leukocytes from the periphery continuously enter the thymic medulla of adult rats: phenotypes, sources and magnitude of traffic

Although the thymus is primarily noted for the export of T cells to the periphery, a small influx of cells has also been observed. It is still a matter of debate whether entry into the thymus depends on prior activation. The phenotypes, sources and degree of immigration are largely unknown. We monitored by quantitative immunohistochemistry the entry of cells from the periphery into the rat thymus in three experimental models. We injected i.v. recirculating, small, nonactivated CD4+ T cell subsets, often referred to as naive (CD45RC+) and memory or antigen-experienced (CD45RC-) cells, purified from thoracic duct lymph of allotype-marked donors, allotype-marked leukocytes released from spleen or lung transplants, or leukocytes labeled in the periphery for 12 weeks during the S-phase of the cell cycle by oral application of 5-bromo-2-deoxyuridine (BrdUrd). Early after i.v. injection (0.5 h), significantly more antigen-experienced (CD45RC-) CD4+ T cells entered the thymus, and by 24 h four times as many cells from the CD45RC- subset as from the CD45RC+ subset had entered the thymus and localized to the medulla. None of the thymic entrants expressed the interleukin (IL)-2 receptor. Following spleen transplantation approximately 40% of donor cells entering the thymic medulla were T cells and approximately 55% were B cells. In contrast, from a lung transplant, approximately 85% of peripheral immigrants were T cells and approximately 10% were B cells. After both procedures, a small number of NK cells and monocytes/macrophages were found among the immigrants (< 5%). Rats were fed BrdUrd continuously for 12 weeks, a procedure which labeled approximately 30% of peripheral lymphocytes but not cortical thymocytes. BrdUrd-labeled cells were localized almost exclusively to the thymic medulla and represented approximately 10% of medullary cells. Of the thymic immigrants approximately 50% were T cells, approximately 30% were B cells (including approximately 15% IgD+ cells), approximately 15% were NK cells and the remainder (approximately 5%) were monocytes/macrophages. Only a quarter of BrdUrd-labeled cells expressed the IL-2 receptor. The thymus is continuously infiltrated by both activated and nonactivated leukocytes from the periphery, including T cells, B cells, NK cells and monocytes. These immigrants are supplied by lymphoid and nonlymphoid organs in a characteristic subset composition. Their entry is facilitated by prior antigen experience or activation. Thus, the participation of the thymic medulla in general leukocyte traffic suggests a mechanism by which the T cell repertoire could potentially be modulated by the peripheral tissues.     

Analysis of type 1 and type 2 T cells in synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: It has been reported that CD4+ helper T cells play an important role in the pathogenesis of rheumatoid arthritis (RA). We evaluated the presence of intracellular cytokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) produced by CD4+ and CD8+ T cells in the synovial fluid and peripheral blood of patients with RA at the single cell level. METHODS: We used 3 color flow cytometric analysis. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were stimulated with phorbol myristate acetate (PMA) and calcium ionophore. The stimulated SFMC and PBMC were triple stained with conjugated mononuclear antibodies (Mab) against cytokines and surface antigens after fixation and permeabilization with a saponine buffer solution. The cells were analyzed for intracellular cytokines (IFN-gamma, IL-4) and surface antigens (CD3, CD4, CD8) using a flow cytometer. RESULTS: The CD4/CD8 ratio was significantly lower in SFMC than in PBMC. The positive rates of IFN-gamma producing cells among CD4+ T cells were significantly higher than those of IL-4 producing cells in both the SFMC and the PBMC of patients with active RA. In the SF of these patients, we also found CD8+ T cells that produce IL-4 alone, or both IL-4 and IFN-gamma. CONCLUSION: In the SF of patients with RA, CD4+ type 1 T cells, which may infiltrate into the synovium and cause pathogenic immune responses in the tissue, are predominant. We believe this cell type also induces migration and activation of CD8+ type 2 T cells into the active site of inflammation, which appears to downregulate the activity of CD4+ type 1 T cells, modulating the excess immune response.     

Characterization of two second messenger pathways and their interactions in eliciting the human sperm acrosome reaction

Actinobacillus actinomycetemcomitans, a gram-negative, capnophilic bacterium, is associated with several human diseases and is the suspected etiologic agent in certain forms of periodontal disease. We have previously shown that this organism produces an immunosuppressive factor (ISF) which is capable of inhibiting both T- and B-cell activation. Furthermore, these effects appear to be associated with the activation of a population of suppressor cells. We now report that the ISF induces a unique population of CD4+CD8+ dual-positive T-cells. By utilizing multiparameter flow cytometric analysis, we were able to detect the presence of dual-positive cells in cultures of human T-cells treated with PHA and ISF. The cells appeared within 48 hr and their induction was dependent upon the presence of both CD4 and CD8 cells in the culture. Dual expression of CD4 and CD8 was stable in that the cells continued to express both surface proteins after being sorted and cultured for an additional 24 hr. Phenotypic analysis indicates that these cells are also CD3+, CD2+, CD5+, TCR alpha beta+, CD45RA+ (and RO+), and CD29+. The dual-positive cells express surface markers associated with T-cell activation: CD25+, CD69+, CD71+, and HLA-DR+. In contrast, the cells were negative for CD34, CD57, CD56, and CD16. Cell cycle analysis indicates that > 80% of the dual-positive cells were in the S phase. Finally, functional analysis of these cells indicates that they are capable of suppressing the proliferative response of autologous T-cells to PHA.