Illness is reduced in mice treated with anti-LFA-1.
BALB/c mice received 200 μg of an isotype control antibody or anti-LFA-1 antibody intraperitoneally at days −1, +1, and +4 relative to the time of RSV infection. On day 0, mice were infected intranasally with 107 PFU of RSV in 100 μl of 10% EMEM. Mice treated with the isotype control exhibited a typical pattern of RSV-induced illness. Peak weight loss was about 18% of original weight on day 7 postinfection (Fig. ). In contrast, peak weight loss in mice that received anti-LFA-1 treatment was only 4% of original weight on days 8 to 9 postinfection. Clinical illness score patterns paralleled the weight loss data in both groups (Fig. ). The peak illness occurred earlier in isotype control-treated mice and was more severe. These illness profiles suggest that anti-LFA-1 treatment offers some protection against RSV-induced illness.
FIG. 1. Illness profiles of mice treated with anti-LFA-1 during primary RSV infection. Mice were infected intranasally with 107 PFU of RSV on day 0. On days −1, +1, and +4 relative to infection, 200 μg of isotype control antibody (more ...) Viral clearance from the lungs is delayed during primary RSV infection in anti-LFA-1-treated mice.
RSV titers were measured in the lungs on days 4, 6, 8, 10, and 12 postinfection (Fig. ). In the lungs, viral titers on day 4 showed no significant difference between the control group and the anti-LFA-1-treated group (P > 0.05). On days 6 and 8 postinfection, mice treated with anti-LFA-1 retained significantly more virus than the isotype control-treated mice (P < 0.05). By day 8, RSV could not be detected in the lungs of isotype control-treated mice but was still present in the lungs of anti-LFA-1-treated mice. By day 10 postinfection, virus had been cleared in both groups. These data indicate that treatment with anti-LFA-1 results in a delay in viral clearance.
FIG. 2. RSV titers in lungs of anti-LFA-1-treated mice. RSV-infected mice were treated with isotype control or anti-LFA-1 antibody as previously described. On days 4, 6, 8, 10, and 12 postinfection, lungs were harvested from RSV-infected mice and viral titers (more ...) Anti-LFA-1 therapy impairs CTL-mediated killing.
Since anti-LFA-1 treatment caused a delay in viral clearance, we reasoned that the cytolytic activity of CTLs was impaired in these mice. To this end, lungs were harvested on day 8 postinfection from RSV-infected mice that received 200 μg of either isotype control or anti-LFA-1 antibody at days −1, +1, and +4 relative to the time of RSV infection. We chose to examine the cytolytic effector function of virus-specific CTLs on day 8 because that is the day of peak CD8+ T cell-mediated cytolytic activity in response to primary infection. Cytolytic activity of these lymphocytes was measured by incubation with a persistently RSV-infected mouse fibroblast cell line, BCH4, in a direct 51Cr release assay (Fig. ). A high percentage of RSV-specific CTL activity was observed in isotype control-treated mice. In contrast, lymphocytes from mice treated with anti-LFA-1 demonstrated a sixfold lower effector activity at the same dilution. No specific lysis was observed when CTLs from either group were incubated with an uninfected fibroblast cell line. From this result, we conclude that anti-LFA-1 treatment significantly impairs the lytic activity of RSV-specific CTLs.
FIG. 3. RSV-specific cytolytic activity. RSV-infected mice were treated with isotype control or anti-LFA-1 antibody as previously described. On day 8 postinfection, lungs were harvested to determine the cytolytic activity of lung lymphocytes by the 51Cr release (more ...) Anti-LFA-1 treatment delays the kinetics of the CTL response during primary RSV infection.
The binding of LFA-1 to ICAM-1 facilitates lymphocyte migration, as well as adhesion and costimulation during antigen presentation. Since our previous data indicated a delay in the immune response during anti-LFA-1 treatment, we hypothesized that extending the treatment with anti-LFA-1 would further delay the RSV-specific immune response. To test this, we produced a primary RSV infection in three additional groups of mice. Two independent groups of mice were treated with either the isotype control or anti-LFA-1 at days −1, +1, and +4, exactly as in our previous experiments. In addition, we administered anti-LFA-1 antibody to a third group of mice on days −1, +1, +4, and +7. Mice were sacrificed at days 6, 8, 10, and 12, and the right lungs were used in ICS for IFN-γ to define the effect of LFA-1 on CTL function during primary RSV infection.
Prolonged treatment with anti-LFA-1 further delayed detection of functional CTLs at the site of infection, as evidenced by a reduction in the percentage of IFN-γ-producing CD8+ T cells (Fig. ). In isotype control-treated mice, peak CTL activation was observed on day 6 postinfection, when approximately 5% of CD8+ T cells produced IFN-γ. IFN-γ production by CD8+ T cells then steadily declined for the duration of the experiment, indicative of a normal pattern of CTL activation during RSV infection. In mice that received anti-LFA-1 through day 4, CTL production of IFN-γ was delayed, steadily increasing until it peaked on day 10. A similar pattern of delayed IFN-γ production was seen in mice that received anti-LFA-1 through day 7. When we examined IFN-γ production by cytokine ELISA of lung supernatants from these mice, we observed the same trends (Fig. ). The peaks of IFN-γ production were therefore not only delayed but also reduced in both anti-LFA-1-treated groups, with production slightly more depressed in the mice that received prolonged anti-LFA-1 treatment. These results suggest that anti-LFA-1 treatment delays the appearance of functionally active CD8+ T cells responding to the viral infection.
FIG. 4. Kinetics of IFN-γ production by CD8+ T cells in anti-LFA-treated mice. Mice were treated with isotype control antibody or anti-LFA-1 antibody on days −1, +1, and +4 (d4) relative to RSV infection. A third group (more ...) Anti-LFA-1 treatment impairs lymphocyte activation.
LFA-1 is known to contribute to lymphocyte migration as well as play a potential role in activation. For this reason, we chose to examine the effect of anti-LFA-1 treatment on the pulmonary lymphocytic infiltrate during RSV infection. The absolute number of CD8+ T cells was lower in the lungs of anti-LFA-1-treated mice at the earliest time points (Fig. ), but by the conclusion of the experiment, CD8+-T-cell numbers were equivalent to those in isotype control-treated mice. These data suggested that anti-LFA-1 treatment might also be inhibiting CD8+-T-cell migration to the lungs. However, delayed activation of CD8+ T cells in the lymph nodes would also preclude their migration into the periphery in search of infected target cells. Consequently, a significant impairment in activation would similarly result in reduced lymphocyte numbers in the lungs. In addition, a late schedule of anti-LFA-1 treatment on days 5, 7, and 10 postinfection had no effect on illness, viral clearance (Fig. ), or cytolytic activity. In addition, no significant difference in lung pathology or degree of inflammatory infiltrate was noted in mice starting treatment on day 5 (data not shown). Treatment on day 5 begins after activation has been initiated but before activated CTLs begin to infiltrate the lungs. The results of this experiment strongly argue against anti-LFA-1 treatment causing a significant effect on lymphocyte migration. We therefore focused on the role of impaired activation as the potential mechanism for the effect.
FIG. 5. Lymphocyte numbers in lungs of anti-LFA-1-treated mice. The absolute number of CD8+ T lymphocytes in the lungs of the mice used for the experiments described in the legend to Fig. was determined by flow cytometry. The results shown (more ...)
FIG. 6. Illness and virus clearance in mice with delayed anti-LFA-1 treatment during primary RSV infection. Mice were infected as previously described. On days +5, +7, and +10 relative to infection, 200 μg of isotype control antibody (more ...)
The impact of anti-LFA-1 treatment on lymphocyte proliferation was evaluated both in vivo and in vitro. First, we treated mice with BrdU to examine lymphocyte proliferation during anti-LFA-1 treatment in the context of primary RSV infection. We used two groups each of isotype control-treated mice and anti-LFA-1-treated mice. The first pair of groups received a single daily intraperitoneal injection of 100 μg of BrdU on days 0 to 5 postinfection, and the second pair of groups received BrdU on days 5 to 9. The staggering of the BrdU treatments was performed to determine the timing of CTL activation and expansion. All mice received 200 μg of either isotype control or anti-LFA-1 antibody at days −1, +1, and +4 relative to the time of RSV infection. We observed that proliferation of CD8+ T cells occurred earlier in the isotype control-treated mice (Fig. ). Isotype control-treated mice were characterized by a dramatic reduction in proliferation between days 8 and 12. In contrast, proliferation of CD8+ T cells increased in anti-LFA-1-treated mice between days 8 and 12. Moreover, the level of proliferation in anti-LFA-1-treated mice was not as robust as that observed in isotype control-treated mice. These data indicate that the administration of anti-LFA-1 to RSV-infected mice resulted in a delayed and inefficient activation and expansion of CD8+ T lymphocytes.
FIG. 7. Lymphocyte proliferation during anti-LFA-1 treatment. (A) RSV-infected mice were treated with either isotype control antibody or anti-LFA-1 antibody as previously described. One set of six mice from each group was then treated intraperitoneally with 100 (more ...)
To better understand the timing of the anti-LFA-1 effect, we performed additional in vitro studies. We isolated lymphocytes from the spleens of RSV-immune BALB/c mice, labeled them with CFSE, and incubated them in the presence of 500 μg of either the isotype control antibody or anti-LFA-1. The lymphocytes were also supplemented with costimulatory antibodies, IL-2, and the immunodominant RSV M2 epitope for 5 days at 37°C. When we examined the cells by flow cytometry, it was very clear that anti-LFA-1 compromised lymphocyte proliferation (Fig. ). Isotype control-treated lymphocytes that were stimulated with the RSV peptide exhibited a maximum proliferation of 4.13% ± 2.26%, as determined by CFSE dilution. In contrast, proliferation of anti-LFA-1-treated lymphocytes was only 0.07% ± 0.09%. The cumulative results of these experiments confirm that anti-LFA-1 treatment impairs lymphocyte activation by interfering with an early step of the antigen presentation process.
Lung pathology in mice treated with anti-LFA-1 is similar to that in control mice.
Since a significant portion of the illness observed during primary RSV infection is immune mediated, anti-LFA-1 has the effect of reducing illness, as shown in Fig. . However, it also leads to delayed clearance of the virus. We therefore examined pathology in the lungs of isotype control (Fig. ) - and anti-LFA-1 (Fig. )-treated mice on day 8 postinfection, the day of peak cytolytic activity. The overall level of perivascular and peribronchiolar infiltration was similar in anti-LFA-1-treated mice and isotype-treated controls. However, the anti-LFA-1-treated mice had a more diverse cell population in the infiltrate that included a higher frequency of macrophages and polymorphonuclear leukocytes. The composition of the infiltrate in the isotype-treated controls was more uniform, with a higher frequency of lymphocytes. This difference in the composition of the infiltrate may reflect a compensatory response to the lack of effective RSV-specific CTLs in the lungs of anti-LFA-1-treated mice.
FIG. 8. Lung histopathology of anti-LFA-1-treated mice. RSV-infected mice received 200 μg of either isotype control or anti-LFA-1 antibody on days −1, +1, and +4 relative to the time of primary RSV infection. On day 8 postinfection, (more ...) Anti-LFA-1 treatment leads to reduced cytokine mRNA levels in the lungs of RSV-infected mice.
The kinetics of viral clearance and CTL induction may be influenced by cytokine expression. For this reason, we performed RPAs to determine cytokine mRNA levels in the lungs of isotype control- and anti-LFA-1-treated mice following primary RSV infection. We examined mRNA production of the cytokines included in the RiboQuant mCK-1 and mCK-2b template sets from BD Pharmingen. No significant differences (P > 0.05) between the groups were detected in levels of IL-1α, IL-1β, IL-12p35, and IFN-γ-inducing factor (Fig. ). However, administration of anti-LFA-1 to RSV-infected mice did result in significant reductions (P < 0.05) in levels of IL-1 receptor antagonist, IL-6, IL-10, IL-12p40, and IFN-γ mRNA (Fig. ). Interestingly, the significant reductions were seen in cytokines that are largely produced by antigen-presenting cells (APC) and lymphocytes after their reciprocal activation following interaction through the immunological synapse. This observation supports the concept that blocking LFA-1 function negatively impacts T lymphocytes by inhibiting the initial APC-T lymphocyte interaction required for activating the expansion and cytolytic function of CD8+ CTLs.
FIG. 9. Cytokine mRNA levels after RSV infection of anti-LFA-1-treated mice. RSV-infected mice were treated with isotype control or anti-LFA-1 antibody as previously described. Four days postinfection, induction of cytokine mRNA was examined by RPA by using radiolabeled (more ...) The RSV-specific antibody response is unaltered in anti-LFA-1-treated mice.
We next examined the RSV-specific antibody response to primary RSV infection in mice treated with anti-LFA-1. To accomplish this, sera of mice obtained 4 weeks after primary infection were evaluated by measuring the total immunoglobulin to the fusion protein of RSV (F). The titers of F-specific immunoglobulin G antibody after primary infection were similar in both groups (Fig. ). In control mice, the titer was measured as a 14.7 ± 0.29 log2 reciprocal serum dilution, whereas the titer of mice treated with anti-LFA-1 was a 13.9 ± 0.74 log2 reciprocal serum dilution. The similarities in antibody titers after infection indicate that anti-LFA-1 treatment does not affect the humoral immune response to primary RSV infection. Furthermore, when these findings are combined with our other data, it appears that LFA-1 is more important for major histocompatibility complex class I costimulation than for major histocompatibility complex class II stimulation during primary RSV infection and has a more profound effect on CD8+ T cell-mediated events than on CD4+-T-cell function.
FIG. 10. RSV-specific antibody titers. RSV-infected mice were treated with isotype control or anti-LFA-1 antibody as previously described. Serum samples were collected from RSV-infected mice on day 28 postinfection. The total amount of antibody specific for the (more ...)