The immunologic and inflammatory response to sepsis is extremely complex and typified by an early net proinflammatory response followed by immunosuppression (43
). There is increasing recognition that the immunosuppressive phase of sepsis, which has been termed immunoparalysis, is a major contributing factor to its lethality (2
). Patients with sepsis may die of an inability to eradicate their initial infection or by developing secondary nosocomial infections. One of the most important factors in sepsis immunosuppression is the apoptosis-induced depletion of immune effector cells (13
). In addition to numerous animal studies documenting widespread loss of immune cells in sepsis, three autopsy studies of patients who died of sepsis documented massive loss of CD4 T cells, B cells, dendritic cells, and other immune elements (7
). In addition to the loss in critical immune cells, apoptosis is detrimental to host immunity through a second independent mechanism. Phagocytic cells that consume apoptotic cells become anergic and/or induce T cells to acquire a Th2 anti-inflammatory phenotype that can inhibit the ability of the host to eradicate the pathogens (48
). A number of investigative groups, including our own, have demonstrated that prevention of apoptosis improves survival in sepsis, thereby supporting the hypothesis that apoptosis of immune cells is a central pathophysiologic event in the disease (13
). Therefore, development of a clinically applicable therapy that could prevent sepsis-induced apoptosis of immune cells and maintain or enhance immune effector cell function could have a major impact on morbidity and mortality in this disorder that kills >210,000 patients annually.
The present work is significant because it demonstrates that rhIL-7, a potent antiapoptotic cytokine, was able to prevent the loss of CD4 and CD8 T cells as reflected by its effects on absolute cell counts and apoptosis. These protective effects occurred in spleen as well as in mesenteric lymph nodes that are inclose proximity to the septic focus. rhIL-7’s protection extended broadly to most classes of CD4 and CD8 T cells (i.e., naive, central memory, and effector memory subsets) and was persistent throughout the duration of sepsis (Supplemental Fig. 1).
Although IL-7 inhibits apoptosis via several mechanisms, one of its most important antiapoptotic actions is due to its ability to increase Bcl-2 (52
). In the current study, rhIL-7 acted rapidly within 24 h to significantly increase the abundance of intracellular Bcl-2 in CD4 and CD8 T cells. The in vitro qRT-PCR data showed a >10-fold increase in mRNA for Bcl-2 in CD4 T cells by 5 h after treatment with rhIL-7 (). These findings are consistent with work by Sportés et al. (26
), who showed that cancer patients treated with rhIL-7 had a dramatic increase in MFI of Bcl-2 in circulating CD4 and CD8 T cells. The Bcl-2–inducing property of IL-7 is particularly important considering the studies that examined the protective effect of transgenic overexpression of Bcl-2 in sepsis. Three independent groups demonstrated that mice that overexpress Bcl-2 are resistant to sepsis-induced apoptosis and have improved survival (13
A surprising finding was the ability of rhIL-7 to prevent the - sepsis-induced increase in mRNA for proapoptotic BH3 molecules, including Bim, PUMA, and BMF. Previous work from our group 7 showed that Bim-null mice and PUMA-null mice have significant decreases in sepsis-induced lymphocyte apoptosis (35
). The ability of rhIL-7 to decrease PUMA in sham- and CLP-operated mice occurred at 8 and 18 h following in vivo administration, and the effect was observed in isolated CD4 T cells using in vitro studies (). Significantly, this rhIL-7–mediated decrease in mRNA for PUMA was associated with an ~39% decrease in intracellular PUMA protein in CD4 T cells (). An additional antiapoptotic effect of rhIL-7 was its ability to increase mRNA for Bcl-2 at 5 h (); however, this effect was not seen at later time points (Supplemental Fig. 3). Given the increase in Bcl-2 protein (), we speculate that the effect of rhIL-7 on mRNA for Bcl-2 may be time dependent.
The most important finding of the current study was the ability of IL-7 to improve survival in sepsis. The beneficial effects of IL-7 on survival were demonstrated for two different preparations of IL-7 in two different institutions (see Materials and Methods
). rhIL-7 complexed with anti–IL-7 Ab and rhIL-7 from Cytheris (which has a long circulating half-life and does not require Ab stabilization) had salutary effects in sepsis. Recent work showed that complexing rhIL-7 to an anti–IL-7 mAb increases its efficacy by 50- to 100-fold (22
). Exceedingly small amounts of anti–IL-7 Ab dramatically prolong the circulating half-life of IL-7 (22
). Similar effects were shown for IL-2 and -15. The rhIL-7 preparation from Cytheris is the formulation being used in the multiple clinical trials previously discussed; it possesses low immunogenicity and has an excellent safety record in patients (26
A key goal of the current study was to determine potential mechanisms of action for the beneficial effect of rhIL-7 in addition to its ability to block apoptosis. One potential mechanism is the ability of rhIL-7 to reverse the decrease in IFN-γ production that is a characteristic finding in animal and clinical studies in sepsis (39
). Studies examining the amount of IFN-γ produced by CD4 and CD8 splenocytes (i.e., intracellular staining for IFN-γ) showed that CD4 and CD8 T cells from septic mice treated with rhIL-7 had an increase in MFI for IFN-γ compared with septic mice that did not receive rhIL-7 (). These results are consistent with work by Levy et al. (28
), who documented that rhIL-7 increased the median frequency of CD4 T cells producing IFN-γ in HIV-1–infected adults. Animal studies showed that treatments that reverse the defect in IFN-γ production can improve survival in sepsis (39
). Furthermore, Döcke et al. (39
) reported that administration of IFN-γ restored monocyte TNF-α production and improved survival in a group of patients with sepsis. In the current study, splenocytes from septic mice treated with rhIL-7 had a reversal of the sepsis-induced defective production of IFN-γ (). IFN-γ production in sham mice treated with rhIL-7 was not different from sham mice not treated with rhIL-7. This latter finding suggests that rhIL-7 did not prime cells for excessive production, but rather reversed the sepsis-induced defect. One possible explanation for the increase in IFN-γ production in the splenocytes from septic mice treated with rhIL-7 is that splenocytes from rhIL-7–treated mice had improved survival during the overnight incubation and, therefore, produced more IFN-γ. It is also possible that rhIL-7 improved the ability of the individual splenocytes to produce more IFN-γ.
A second potential mechanism for the salutary effect of IL-7 in sepsis is its action to increase expression of the leukocyte adhesion molecules LFA-1 and VLA-4. The effect of IL-7 on integrin expression has been examined to a very limited degree; the present report is the first study to show that IL-7 increases the expression of multiple integrins in an in vivo setting and during a pathologic state. A successful immune response depends on the ability of the immune effector cells to migrate from one location in the body to another (54
). The increase in leukocyte adhesion markers results in improved leukocyte migration and tissue penetration to the site of infection or to other areas in which critical cell cross-talk will occur. Integrins, especially LFA-1, are essential for most adhesion-dependent lymphocyte functions, including Ag- and APC-induced Th cell stimulation, cytotoxic lymphocyte-mediated killing of target cells, and adhesion of lymphocytes to vascular endothelium (53
). Although the role of integrins in sepsis is not well defined, Prince et al. noted that mice genetically deficient in LFA-1 that were challenged with i.p. Streptococcus pneumoniae
had increased bacterial colony counts in spleen and liver, increased incidence of otitis media and meningitis/encephalitis, and increased mortality compared with wild type mice (57
). The increase in LFA-1 and VLA-4 in the current study is probably related to a direct ability of rhIL-7 to increase integrin expression as well as a potential ability of rhIL-7 to prevent apoptosis in cells that are expressing these integrins. A key finding is that rhIL-7 increased LFA-1 and VLA-4 in CD4 and CD8 T cells from sham-operated mice to an even greater extent than in the CLP-operated mice (). Because there will be no increased apoptosis in T cells from sham-operated mice, the ability of rhIL-7 to increase LFA-1 and VLA-4 in sham mice and, therefore, likely in the setting of sepsis as well, is probably a direct effect.
Importantly, rhIL-7 increased central and effector memory CD4 and CD8 T cells in mesenteric lymph nodes of septic mice. Multiple mechanisms likely contribute to this increase in cell number. Certainly, increases in Bcl-2 levels likely provide protection from apoptosis to these cells. In addition, increased Ki-67 staining () indicates that rhIL-7 promotes cellular proliferation. Further, rhIL-7–driven increases in integrin expression could enhance T cell numbers by facilitating lymphocyte trafficking to and from sites of infection. Finally, rhIL-7 promotes the expression of chemokine receptors that promote lymphocyte migration (30
). Beq et al. (30
) recently showed that Rhesus macaques that received rhIL-7 had increased T cell expression of homing chemokine receptors, including CXCR4, CCR6, and CCR9, as well as increased tissue expression of numerous chemokines. This effect of rhIL-7 was associated with massive and rapid T cell migration from the blood into various organs, including lymph nodes and parts of the intestine. One intriguing possibility is that rhIL-7 promotes lymphocyte viability and, in doing so, allows them to respond to signals (i.e., chemokines) that let them increase the expression of integrins, migrate to sites of infection, and promote pathogen elimination.
In addition to IL-7’s ability to increase integrin expression, the present work demonstrates that it also increased the MFI of CD8 by ~50.3% ± 3.5% and 29.3% ± 3.2% on individual cytotoxic T cells in sham and CLP mice, respectively (). CD8 is a coreceptor for MHC class I molecules and binds Lck on the cytoplasmic face of the plasma membrane. The increased expression of CD8 and integrins induced by IL-7 likely leads to more sustained interactions at the immunologic synapse between the CD8 TCR and MHC class I and, thereby, improves cytotoxic T cell activation. The present findings represent the first report that IL-7 increases CD8 expression in a clinical disorder and are consistent with the one prior study on this topic by Park et al., who observed that signals from IL-7 and other common γ-chain cytokines transcriptionally increase CD8 coreceptor expression in individual CD8 T cells (58
In addition to improving survival, the most significant effect of rhIL-7isitsabilitytorestore the DTH response insepsis. Patients with sepsis lose their DTH response to recall Ags, a finding that is emblematic of their severely impaired immune function (59
). In addition to the preservation of functional T cells, a potential mechanism for the improved DTH response in septic mice treated with rhIL-7 may be IL-7’s reported ability to enhance T cell–APC interactions (20
). The present work is consistent with studies by Fry et al. (42
), who determined that IL-7 restored the ability of athymic T cell-depleted hosts that underwent adoptive transfer of lymph node cells to appropriately reject nonmatched skin grafts. They stated that the restoration of immune competence seemed to be mediated by a combination of inhibition of apoptosis, enhanced costimulation, and modulation of APC function.
In addition to its antiapoptotic effects, rhIL-7 acts to restore CD8 T cells that are depleted during sepsis by its ability to induce proliferation (25
). Geiselhart et al. (25
) showed that a 2-d administration of IL-7 in vivo increased basal proliferation of CD4 and CD8 T cells by 4- and 18-fold, respectively. In a recent National Cancer Institute study of 16 patients with refractory cancer, patients who received the highest doses of rhIL-7 had a >60% increase in spleen size and an ~2- and 4-fold increase in circulating CD4 T and CD8 T cells, respectively (26
). These effects of rhIL-7 occurred within 14–28 d of therapy. Additional studies showed that >40% of CD4 T cells and >50% of CD8 T cells were positive for Ki67, a marker of cell proliferation. In animal studies, the ability of IL-7 to induce proliferation of T cells starts to occur within 48–72 h. In the current study, rhIL-7 caused an approximate doubling in CD8 T cell proliferation in septic mice at 72 h after administration of rhIL-7 ().
A not unexpected discovery was the increase in IL-7R expression on CD4 and CD8 T cells from septic mice compared with sham-operated mice (). As noted in numerous studies (14
), as well as documented in the current study (, ), sepsis induces massive loss of immune effector cells, including CD4 and CD8 T cells. This loss in immune cells triggers homeostatic proliferative mechanisms that ultimately result in replenishment of tissue stores of CD4 and CD8 T cells in surviving animals. IL-7 is critical for a proper homeostatic proliferative response, and the increased IL-7R expression in CD4 and CD8 T cells likely serves to enhance IL-7’s effect (20
). T cells from septic mice had markedly upregulated IL-7Rs (CD127), and septic mice had an increase in circulating IL-7 compared with sham mice. Research indicates that the amount of IL-7R expression on a cell determines how vigorously the cell responds to IL-7 (31
). Previous work from our group showed that sepsis inhibits homeostatic proliferation in CD4 T cells (33
); the ability of rhIL-7 to increase proliferation in CD8 T cells from septic mice () may be advantageous in overcoming this inhibition.
A theoretical advantage of the use of rhIL-7 in sepsis is that it is unlikely to exacerbate the initial hyperinflammatory phase that usually precedes the more sustained secondary hypoinflammatory stage of sepsis (26
). The early phase of sepsis is often characterized by a proinflammatory cytokine-mediated syndrome of fever, hypotension or shock, and acute lung injury (2
). The administration of agents that accentuate the inflammation may worsen early mortality in sepsis (62
). Unlike IL-2, a closely related cytokine, IL-7 rarely induces fever, capillary leak syndrome, or other clinical abnormalities related to the release of excessive proinflammatory cytokines (26
). Studies show that IL-7 can induce proliferation in the absence of cytokine production and without increases in most lymphocyte-activation markers (22
). One potential explanation for the fact that IL-7 does not exacerbate inflammation is related to the fact that as T cells become activated, there is downregulation of IL-7R expression (31
). This down-regulation of the receptor likely helps to prevent hyperactivation of the T cells. We speculate that rhIL-7 may preserve the potential for effector function of T cells without causing outright activation. This speculation is supported by the results of the splenocyte-incubation studies in which rhIL-7 reversed the defect in sepsis-inducted IFN-γ production but did not increase IFN-γ production in the sham treated-mice.
In conclusion, although there are a number of potentially promising therapies in sepsis, we speculate that rhIL-7 is particularly attractive because it ameliorates many of the key pathophysiologic processes that are believed to be central to the lethality of sepsis. Specifically, rhIL-7 blocks sepsis-induced depletion of CD4 and CD8 T cells, enhances lymphocyte recruitment, prevents the sepsis-induced loss in immunity as evidenced by preserved DTH response, does not exacerbate the proinflammatory response in sepsis, and improves survival. If current clinical trials of rhIL-7 continue to demonstrate its safety in multiple patient populations, rhIL-7 could move rapidly into clinical trials in sepsis.