Active immunization by a dengue virus-induced cytokine.

Dengue type 2 virus (DV)-induced cytotoxic factor (CF) is capable of reproducing various pathological lesions in mice that are seen in human dengue. The present study was undertaken to investigate the protective effect of active immunization of mice with CF. Mice were immunized with 5 microgram of CF and prevention of CF-induced increase in capillary permeability and damage to the blood-brain barrier were studied at weekly intervals, up to 48 weeks, by challenging with 3 microgram of CF. Maximum protection against increase in capillary permeability and damage to the blood-brain barrier was observed in week 4 after immunization. A breakthrough in the protection occurred with higher doses of CF in a dose-dependent manner. Challenge with a lethal intracerebral (i.c.) dose of DV showed significantly prolonged mean survival time and delayed onset of symptoms of sickness in the immunized mice compared with the normal mice, but the titre of the virus in the brain was similar in the two groups. On i.p. challenge with the virus the protection against damage to the blood-brain barrier was 86 +/- 7% at week 4 and 17 +/- 4% at week 26 after immunization. Sera obtained from the immunized mice showed the presence of CF-specific antibodies by ELISA, Western blot, and by neutralization of the cytotoxic activity of CF in vitro. The present study describes successful prevention of a cytokine-induced pathology by specific active immunization.     

Transmission of dengue virus-induced helper signal to B cell via macrophages.

The helper T cells (TH) generated in dengue type 2 virus (DV) infection of mice produce a soluble helper cytokine (HF) which enhances the clonal expansion of DV-specific IgM antibody plaque forming cells (PFC). The present study was undertaken to investigate the mechanism of transmission of the helper signal from TH and HF to B cells. It was observed that TH could transmit the helper signal to B cells by direct cell to cell contact, but HF could not do so without the presence of live macrophages (M phi). HF was adsorbed by both heat killed and live M phi but the former could not transmit it to B cells. Both the polypeptide chains of HF bind to M phi. HF remains on the surface of M phi and can be retrieved completely by contact with B cells for 40 min. The helper signal from TH or HF-adsorbed M phi could not be transmitted to B cells when they were separated from each other by a cell impermeable membrane. The enhancement of PFC count is greater when the signal is transmitted by HF-adsorbed M phi as compared to that by TH alone. Thus, even with lower frequency of TH a significant number of B cells may be triggered with the help of HF and M phi. The findings thus show that the DV-specific helper signal could be transmitted only by a close physical contact of the plasma membranes of the signal presenting cells (TH or HF-adsorbed M phi) and B cells.     

Dengue virus-induced helper cytokine has two polypeptide chains which bear different determinants.

Dengue type 2 virus (DV) induces generation of a T cell helper cytokine (HF) in mouse spleen which enhances the antigen-specific antibody plaque forming cell count in syngeneic mice. The present study was undertaken to investigate the molecular structure of HF. It was observed that the activity of HF was abrogated by treatment with reducing agents such as glutathione, ouabain or dithiothreitol (DTT) which cleave disulphide bonds, separating the polypeptide chains. The two polypeptide chains could be purified by high performance liquid chromatography of DTT treated HF. The individual chains had no helper activity, but it could be restored by mixing the two. One chain of the HF bonded to the DV-antigen coupled immunosorbent column and the other to the anti-I-Ak antibody coupled column. Thus, DV-induced HF is a disulphide bonded double chain structure, one chain having antigen and the other having I-A determinants; the presence of both chains is essential for helper activity.     

Identification and purification of a receptor on macrophages for the dengue virus-induced suppressor cytokine.

Dengue type 2 virus (DV)-induced suppressor cytokine (SF) binds to macrophages to transmit the suppressor signal to recruit the second subpopulation of suppressor T cells. The present study was undertaken to identify and purify the receptor for SF (SF-R) on macrophages. The binding of 125I-SF to macrophages was saturable and reversible. Scatchard analysis showed the presence of both high (54,000/cell) and low (1.78 x 10(6)/cell) affinity receptor sites. The binding of 125I-SF to macrophages was inhibited by pretreatment of macrophages with anti-SF antiserum but not by a heterologous antiserum. Normal mouse peritoneal macrophage membrane was solubilized with Triton-X-100 and the components separated by low pressure liquid chromatography (LPLC) to purify SF-R. The presence of SF binding moiety (SF-R) was screened at each step of purification. The purified SF-R resolved into two bands of 45-50 kD mol. wt on SDS-PAGE. 125I-SF+SF-R complex run on SDS-PAGE showed a single band at about 55-60 kD mol. wt by autoradiography. Anti-SF-R antiserum reacted with SF-R in a Western blot test; the reaction was abolished by pretreatment of the blots with proteinase K, but not by pretreatment with periodic acid. SF-R was composed of two polypeptide chains (alpha and beta) which were obtained in pure form by high performance liquid chromatography (HPLC) of dithiothreitol- and iodoacetamide-treated SF-R. Only the beta chain bound SF.     

Characterization of the cytotoxin produced by macrophages in response to dengue virus-induced cytotoxic factor.

We have observed earlier that dengue type 2 virus-induced cytotoxic factor (CF) induces macrophages to produce a cytotoxin (CF2) which kills mainly the macrophages, some of the T lymphocytes and has no effect on B-lymphocytes of normal mouse spleen. The findings of the present study show that CF2 is heat-labile, trypsin-sensitive and unstable at acid and alkaline pH. It is a low molecular weight product as it is dialysable, non-sedimentable on ultracentrifugation at 103,500 g for 3 h and passes through 0.22 micron Millipore filter. It is adsorbed onto the target normal mouse spleen cells. The properties of CF and CF2 have been compared.     

Production of dengue virus-induced macrophage cytotoxin in vivo.

We have observed that dengue virus-induced cytotoxic factor (CF) induces peritoneal and splenic macrophages in vitro to produce a cytotoxin (CF2). This study demonstrates also production of CF2 in vivo in DV-infected mice and following inoculation with CF. The cell-type responsible for CF2 production in vivo is the macrophage (M phi) as M phi-depleted mice failed to produce CF2. CF2 activity could not be observed in the serum or peritoneal fluid though it is produced in peritoneal M phi. Once stimulated, CF2 is present for 4 h in M phi. M phi can be restimulated to produce CF2 only after a refractory period of 48 h.     

Effect of dengue virus-induced cytotoxin on capillary permeability.

Capillary permeability is increased in cases of dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) but its genesis is not known. Dengue type 2 virus (DV) induces production of a cytokine (CF2) by mouse macrophages. The present study was undertaken to investigate the effect of CF2 on capillary permeability. It was observed that intraperitoneal inoculation of CF2 in mice increased the capillary permeability in a dose-dependent manner, as shown by leakage of intravenously injected radioactive iodine (125I) or Evan''s blue dye in the peritoneal cavity. Peak leakage occurred at 30 min and the vascular integrity was restored by 1-2 h. The increase in capillary permeability was abrogated by pretreatment of mice with avil (H1 receptor blocker) but not by ranitidine (H2 receptor blocker). The findings thus show that DV-induced CF2 increases the capillary permeability via release of histamine.     

Duration of adoptively transferred dengue virus-induced suppressor activity.

Dengue virus-induced suppressor pathway involves three sequential generations of suppressor T cells and their products. The present study was undertaken to determine the duration of suppressor activity of the different components of the suppressor pathway on adoptive transfer in recipient mice. It was observed that the suppressor activity of TS1 cells lasts for 21 days, of TS2 for 15 days, of SF for 7 to 10 days and of SF2 for 7 days. The duration of suppression appears to depend upon the length of suppressor pathway to be followed by each component.     

Purification and amino-terminal sequence of the dengue virus-induced cytotoxic factor.

The present study was undertaken to purify the dengue type 2 virus (DV)-induced cytotoxic factor (CF) and analyse its amino-terminal sequence. Spleens collected from DV-infected moribund mice were made into a single cell suspension and cultured for 24 h. The culture supernatant was purified using fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis (PAGE). CF could be purified in a single step by FPLC and native PAGE. The isoelectric point of CF was pH 6.5. Purified CF had a molecular weight of 45 kDa on native-PAGE while on SDS-PAGE it dissociated into two bands of 20 and 25 kDa. Anti-CF-antisera reacted specifically with CF bands separated on native and SDS-PAGE in a Western blot assay. A sequence of 19 amino-acids of the N-terminus of CF was analysed which on comparison with that of other known cytotoxic proteins indicated that CF differs from them. Thus, CF appears to be a unique cytotoxic protein induced by a virus.     

Study of the target cell of the dengue virus-induced suppressor signal.

Dengue type 2 virus (DV) induces two generations of T suppressor cells (Ts1 and Ts2) in murine spleen. The Ts2 cells produce a soluble factor ( SF2 ) which suppresses DV-specific IgM antibody plaque-forming cells (PFC). In this study the target cell of SF2 was examined. The results show that SF2 requires a third generation of T cells (T3) in order to mediate suppression. The surface phenotype of this T3 cell is Ly 1+ and the suppression mediated is antigen specific.     

Increased capillary permeability mediated by a dengue virus-induced lymphokine.

The mechanism of increased capillary permeability, seen in cases of dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), is not known. Dengue type 2 virus (DV) is known to induce production of a lymphokine, the cytotoxic factor (CF), by the T lymphocytes of mouse spleen. The data presented here show that intraperitoneal inoculation of CF in mice results in increased capillary permeability in a dose-dependent manner, as shown by leakage of intravenously injected radiolabelled iodine (125I) or Evans blue dye. Peak leakage occurred 30 min after inoculation of CF and the vascular integrity was restored by 2 hr. The increase in capillary permeability was abrogated by pretreatment of mice with anti-CF antibodies, avil (H1 receptor blocker) or ranitidine (H2 receptor blocker). The findings thus show that a DV-induced lymphokine, the CF, increases the capillary permeability via release of histamine.     

Effect of adjuvants on immunization with dengue virus-induced cytotoxic factor.

Specific active immunization with dengue type 2 virus (DV)-induced cytokine, cytotoxic factor (CF), prevents CF-mediated pathology in mice. The present study was undertaken to determine the optimum dose of CF and the effect of different adjuvants on the immune response as assessed by the study of anti-CF antibody titre by ELISA and protection against increase in capillary permeability to challenging dose of 3 micrograms CF. The maximum protection of 94 +/- 4% against increase in capillary permeability was observed at week 4 after immunization with 5 micrograms dose of CF mixed with Freund's incomplete adjuvant (FIA), which gradually decreased to 21 +/- 10% on week 24. With a dose of 10 micrograms the protection obtained was 79 +/- 5%, but persisted for a longer time at a higher level. The response was poor with 1 microgram dose of CF. The mean anti-CF antibody titres gradually decreased after reaching the peak at week 4 after immunization. Mice immunized with different adjuvants emulsified with 5 micrograms CF were challenged at different intervals with 3 micrograms CF. Maximum protection observed with CF + tetanus toxoid (TT) and 84/246 was about 93 +/- 2% and 97 +/- 2%, while that with alhydrogel was 33 +/- 12% and with bacille Calmette-Guérin (BCG) was 67 +/- 4%. At week 24 after immunization, however, the best response was obtained with 10 micrograms of adjuvant 84/246. Intracerebral challenge with 10 or 100 LD50 dose of dengue type 2 virus showed significantly prolonged mean survival time and delayed onset of signs of sickness in immunized mice compared with normal mice. The maximum survival time was with adjuvant 84/246 even at week 24. The findings thus show that the optimum dose of CF is 5 micrograms and the adjuvant of choice is 84/246.     

Differential cyclophosphamide sensitivity of T lymphocytes of the dengue virus-induced suppressor pathway.

Dengue type 2 virus (DV) induces a suppressor pathway in mice which involves sequential participation of three subpopulations of T lymphocytes viz. Ts1, Ts2 and T3 cells. In the present study cyclophosphamide (CY) sensitivity of these cells have been investigated. The findings of the present study demonstrate that Ts1 and Ts2 cells are CY-insensitive while T3 cells are CY-sensitive. This supports our earlier conclusion that T3 may be the inducer cells of suppression.     

Production of dengue virus-induced macrophage cytotoxin in vivo

We have observed that dengue virus-induced cytotoxic factor (CF) induces peritoneal and splenic macrophages in vitro to produce a cytotoxin (CF2). This study demonstrates also production of CF2 in vivo in DV-infected mice and following inoculation with CF. The cell-type responsible for CF2 production in vivo is the macrophage (M phi) as M phi-depleted mice failed to produce CF2. CF2 activity could not be observed in the serum or peritoneal fluid though it is produced in peritoneal M phi. Once stimulated, CF2 is present for 4 h in M phi. M phi can be restimulated to produce CF2 only after a refractory period of 48 h.     

Production of nitrite by dengue virus-induced cytotoxic factor

Dengue type 2 virus (DV) infection induces production of a cytokine, the cytotoxic factor (CF) in the spleen of mice. The present study was undertaken to investigate the production of nitrite (NO⁻₂) by the spleen cells of mice in vitro and in vivo following inoculation of DV or CF. Maximum NO⁻₂ production occurred at 45 min after inoculation of 5 μg CF, both in vitro and in vivo. The NO⁻₂ was produced by macrophages and T cells and not by B cells. Pretreatment of CF with anti-CF antisera inhibited production of NO⁻₂. DV-stimulated spleen cell culture supernatants showed peak production of CF and NO⁻₂ at 72 h. In DV-infected mouse spleen, maximum NO⁻₂ production occurred at 8–11 days post-infection, which correlated with peak cytotoxic activity in the spleen. Pretreatment of spleen cells with N^G-monomethyl l-arginine (NMMA) inhibited NO⁻₂ production. NO⁻₂ production was abrogated in a dose-dependent manner by treatment of spleen cells with Ca²⁺ channel blocking drug, Nifedipine. The findings demonstrate that DV-induced CF induces production of NO⁻₂ in spleen cells, probably in a Ca²⁺-dependent manner, and may be a mechanism of target cell killing.     

Characterization of the immunosuppression accompanying virus-induced avian osteopetrosis.

Infection of chickens with a myeloblastosis-associated virus which induced a high incidence of osteopetrosis was accompanied by immunosuppression. The immunosuppression was manifested in the following ways. The weight of the bursa, spleen, and thymus was depressed in infected chickens. Infected animals had a diminished capacity to form hemolytic plaques in a direct assay. Spleen cells from osteopetrotic animals did not respond to phytohemagglutinin, and the spleen and bursa had a decreased proportion of cells possessing surface immunoglobulin. Osteopetrotic animals failed to show an age-dependent increase in the proportion of cells demonstrating surface immunoglobulin that was observed in normal animals. However, several individual chickens with heavy osteopetrosis responded to antigenic stimulation in a normal fashion, indicating that although immunosuppression usually accompanies avian osteopetrosis, it may not contribute directly to abnormal bone proliferation.     

Duration of adoptively transferred dengue virus-induced suppressor activity

Dengue virus-induced suppressor pathway involves three sequential generations of suppressor T cells and their products. The present study was undertaken to determine the duration of suppressor activity of the different components of the suppressor pathway on adoptive transfer in recipient mice. It was observed that the suppressor activity of TS1 cells lasts for 21 days, of TS2 for 15 days, of SF for 7 to 10 days and of SF2 for 7 days. The duration of suppression appears to depend upon the length of suppressor pathway to be followed by each component.     

Effect of dengue virus-induced cytotoxic factor on Fc-receptor functions of mouse macrophages.

Many defects in macrophage functions in dengue type 2 virus (DV) infection have been shown to be mediated by production of the virus-induced cytotoxic factor (CF). An attempt has been made to determine whether the alterations in Fc-mediated functions of macrophages in DV-infected mice are due to CF. Mice given CF intravenously show a rapid fall in total numbers of peritoneal and spleen cells. The number of cells in the peritoneal cavity recovered in 48 h. but recovery in the spleen was not significant. The capacity of the splenic and peritoneal macrophages to attach and ingest opsonized sheep erythrocytes was significantly reduced, the lowest values of attachment index (AI) and phagocytic index (PI) being observed within 2-3 h. At later periods the AI values increased markedly but the PI values remained depressed. The effect was dose-dependent. The effect on Fc-receptor functions of macrophages in DV-infected mice thus appears to be mediated through CF.     

Variable effects of dengue virus-induced cytotoxic factors on different subpopulations of macrophages.

Dengue virus (DV) induces T lymphocytes of the spleen to produce a cytotoxic factor (CF) that induces a subpopulation of macrophages (M phi) to produce a soluble cytotoxin (CF2). Both these factors kill normal lymphoid cells and M phi. The present study was undertaken to investigate the effects of these factors on the I-A-positive and I-A-negative subpopulations of mouse peritoneal M phi. It was observed that CF kills I-A-negative M phi and induces I-A-positive M phi to produce the CF2 that kills both types of cells. However, even when combined together, CF and CF2 do not kill 100% of the M phi. The two-step mechanism involving co-operation between T cells and M phi appears to be biologically economical for maintaining the cytotoxic pathway.     

Obligatory role of Ca2+ in the cytotoxic activity of dengue virus-induced cytotoxin.

The role of calcium ions (Ca2+) in the cytotoxic activity of the dengue type 2 virus (DV)-induced macrophage (M phi) cytotoxin (CF2) was investigated in the present study. The findings show that CF2 prepared in Ca(2+)-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca(2+)-free medium but killed the cells suspended in the medium with Ca2+. Substitution with calcium chloride restored the cytotoxic activity of CF2 the optimal dose being 10(-4) M concentration. CF2 induced an influx of Ca2+, as assayed by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, viz. M phi and T lymphocytes. The cytotoxic activity of CF2 as well as the CF2-induced influx of 45Ca was inhibited by treatment of the target cell with the calcium channel blocking drugs verapamil and nifedipine. Thus, the presence of Ca2+ is obligatory for the cytotoxic activity of CF2 and cell death is associated with increased intracellular Ca2+.