For effective control of viral infections, the bodies of infected persons often mount both adaptive or specific, and innate immune responses (Markine-Goriaynoff, Hulhoven and Cambiaso 2709, par. 1). Cytolytic responses may be initiated by the natural killer (NK) cells or T lymphocytes, but this depends on the infecting pathogen. Other responses to viral infection may be in the form of antibody production, or release of molecules that inhibit replication of the infecting virus. A number of viruses have been able to evade the immune responses and therefore persist in immune-compromised hosts. A good example is the “lactate dehydrogenase-elevating virus (LDV), which persists indefinitely in the circulation of infected mice” (Markine-Goriaynoff, Hulhoven and Cambiaso 2709, par. 1). The LDV virus persists in the mice “despite the production of neutralizing antibodies and the induction of effective helper and cytolytic T lymphocytes” (Markine-Goriainoff, Hulhoven and Cambiaso 2709, par. 2). These immune responses are not effective in controlling viral replication, their specificity not withstanding. However, there is no sufficient data on the innate responses that are triggered by LDV. Responses such as the “NK activation have not been analyzed extensively” (Markine-Goriaynoff, Hulhoven and Cambiaso 2709, par. 4). Therefore, this study was conducted to examine if LDV infection leads to the activation of Natural killer (NK) cells and their resultant IFN-y secretion.
Methods
Pathogen free female mice; “CBA/Ht, DBA/2, BALB/c, BALB/c nu/nu and BALB/cBy-SCID (SCID) and isolator-reared 129/Sv mice were raised at the Ludwig Institute for Cancer Research (Brussels, Belgium) and used when 8±13 weeks old”, Infection by LDV was done via an intra-peritoneal injection (Markine-Goriaynoff, Hulhoven and Cambiaso 2710, par. 1). The monoclonal antibodies for the recognition of the VP3 protein found in the LDV were made available. Suitable antibodies for the inhibition of NK cell activity were prepared in a rabbit using incomplete Freund’s adjuvant and purified asialoganglioside –GMI (Markine-Goriaynoff, Hulhoven and Cambiaso 2710, par. 2). Titration of “LDV was done using the new sensitive particle counting immunoassay based on agglutination of latex beads coated with two different anti-LDV m Abs by virus” particles (Markine-Goriaynoff, Hulhoven and Cambiaso 2710, par. 4). Flow cytometry testing was performed using FACScan flow cytometer which showed the results as a percentage (Markine-Goriaynoff, Hulhoven and Cambiaso 2710, par. 5). The RNA was extracted and gene expression evaluated using RT-PCR protocols.
Results
The effect of LDV on NK cells was initially analyzed by examining the ratio of spleen ad peritoneal cells recognized by DX5 antibody at varying times following infection (Markine-Goriaynoff, Hulhoven and Cambiaso 2711, par. 1). 4 days after the infection with LDV a pronounced rise in the spleen cell population was witnessed in the 129/Sv mice. A similar rise in cell population was also seen in the spleen of BALB/c mice (Markine-Goriaynoff, Hulhoven and Cambiaso 2711, par. 1).
The lytic activity of the spleen and the peritoneal cells from the LDV-infected mice was examined to further investigate NK cell activation following LDV infection (Markine-Goriaynoff, Hulhoven and Cambiaso 2711, par. 2). “NK-sensitive target YAC-1 cells were used to test the lytic activity and against the TEPC.1033 cells” which are known to be resistant to NK mediated lysis (Markine-Goriaynoff, Hulhoven and Cambiaso 2711, par. 2). Significant YAC-1 lysis did not take place in the peritoneal cells that were derived from uninfected mice. However efficient YAC-1 lysis was observed in the cells that were harvested from infected mice. In addition, “TEPC.1033 failed to lyse indicating that the NK cells were the lytic effectors” (Markine-Goriaynoff, Hulhoven and Cambiaso 2711, par. 2). Peak lysis occurred one to two days following mice inoculation with LDV virus. When SCID mice, “which lack ctyolytic T lymphocytes were infected with LDV, the increase in the lytic activity of the peritoneal cells against YAC-1 was also observed” (Markine-Goriaynoff, Hulhoven and Cambiaso 2712, par. 2). Furthermore, the addition of “anti-ASGM1 polyclonal antibody, which usually eliminates NK cells in-vivo, inhibited most of the anti-YAC-1 lytic activity from bulb or SCID” animals infected with LDV (Markine-Goriaynoff, Hulhoven and Cambiaso 2712, par. 2).
The study also revealed that LDV triggered the release of IFN-y gene expression and IFN-y production by NK cells (Markine-Goriaynoff, Hulhoven and Cambiaso 2712, par. 3). The LDV’s effects on NK production were determined by measurements the cytokine level in the mice serum after varying periods of time. The effect was additionally tested by RT-PCR to determine the level of expression. It was established that expression of the gene occurred a short duration after the spleen or peritoneal cells were infected with LDV and peaked in 12 hours. Similar observations were made when “nude mice (treated with depleting anti-CD4 or anti-CD8 mAbs) were used and thus indicating that the message expression and production of this cytokine were independent from T lymphocytes” (Markine-Goriaynoff, Hulhoven and Cambiaso 2712, par. 3). Investigations for the effects NK cells and IFN-y on LDV showed that the two cannot inhibit early LDV replication (Markine-Goriaynoff, Hulhoven and Cambiaso 2712, par. 3).
Discussion
The activation of NK cell and the subsequent cell mediated cytoxicity and IFN-y production is common in many viral infections, “including lymphocytic choriomenengitis virus (LCMV), mouse hepatitis virus (MHV) and murine cytomegalovirus (MCMV)” (Markine-Goriaynoff, Hulhoven and Cambiaso 2713, par. 1). Therefore it does not come as a surprise to observe similar cytolysis following LDV inoculation, although LDV was chiefly known for enhancing humoral immune response while decreasing cellular responses (Markine-Goriaynoff, Hulhoven and Cambiaso 2714, par. 1). This indicates that the increased cytolytic activity following infection with LDV may actually be credited to the virus. Furthermore, the inhibition of the cytolytic effects when anti-ASGM1 antibody is administered indicates that NK cells played a roll in this effect. The results of this study show the diverse processes in the activation different NK cell function triggered LDV inoculation, “with very early IFN-y production rapidly followed by an increase in cytolytic activity and finally a slight delayed of CD49b+ cells” (Markine-Goriaynoff, Hulhoven and Cambiaso 2714, par. 2). Explanation for this occurrence can be linked to the sequential activity facilitated by different cytokines. Cytokines such as the IL-12, IL-15 and growth factor may be responsible for the NK activities as they are usually produced following LDV infection. Previous studies have shown that “LDV varaemia usually persists despite the development of T and B cell mediated anti-viral immune responses and little is known about the effects of NK cells on the replication of this virus” (Markine-Goriaynoff, Hulhoven and Cambiaso 2714, par. 3). Data obtained by this study show that the initial rapid replication phase exhibited LDV virus is not controlled by NK cells. In addition, “the inability of G129 mice to respond to IFN-y did not modify the viral titres” (Markine-Goriaynoff, Hulhoven and Cambiaso 2714, par. 3). This finding validates previous results that showed the inability of IFN-y to inhibit LDV viral replication though it may confer protection against polioencephalomyelitis that is induced by LDV (Markine-Goriaynoff, Hulhoven and Cambiaso 2714, par. 4). In most mouse strains, LDV does not cause any pathological manifestations, however, it affects the immune response leading to enhanced humoral responses are triggered. The virus triggers activation of the immune system to produce the inflammatory responses that are characteristic of its infection (Markine-Goriaynoff, Hulhoven and Cambiaso 2715, par. 1).
Reference
Markine-Goriaynoff, Dominique, et al. “Natural killer cell activation after infection with lactate dehydrogenase-elevating virus.” Journal of General Virology (2002): 83, 2709–2716. Web.