It is widely recognized that sepsis patients are at risk for nosocomial infections, as well as for opportunistic infections usually seen only in immunocompromised patients (21
). The results of clinical and experimental studies have suggested that this might be explained by a biphasic immunological pattern during sepsis: an early hyperinflammatory phase followed by a hypoinflammatory state, the so-called compensatory anti-inflammatory response syndrome (25
). The initial studies of the pathophysiologic events occurring during the hypoinflammatory state of sepsis concentrated on monocyte hyporesponsiveness due to endotoxin tolerance. Endotoxin tolerance consists of a reprogrammed monocyte response to a repeated LPS challenge with respect to the release of proinflammatory cytokines. Monocytes from septic patients have been reported to have a diminished capacity to release TNF-α, IL-1α, IL-1β, IL-6, and IL-12 (9
), whereas unaltered or even enhanced production of anti-inflammatory factors, such as IL-10 and tumor growth factor-β, has been reported (46
). Based on the results of these studies, attempts have been made to restore the systemic proinflammatory cytokine response to endotoxin and/or to neutralize the immunosuppressive effects of anti-inflammatory factors. Both GM-CSF and IFN-γ proved able to restore the LPS responsiveness of monocytes from septic patients (36
). In addition, the ability of human septic plasma to induce tolerance was significantly reduced by anti-IL-10 antibodies (45
). However, none of these therapies were associated with increased bacterial clearance or decreased long-term mortality (36
). Moreover, the results of recent investigations with infectious models using either Cryptococcus neoformans
) or Salmonella enterica
) established that LPS-tolerant mice had an increased resistance to fungal or bacterial infection that was associated with a reduced burden of pathogens within the tissues. It is therefore difficult to assume that endotoxin tolerance per se is directly linked to the increased susceptibility of septic patients to nosocomial infections.
In the quest for immunological alterations that may help explain the impaired immune responses during sepsis, we have explored the possible role of monocyte hyporesponsiveness to CD40L stimulation. We found that for patients with sepsis caused by infection with gram-negative organisms, the ability of monocytes to produce proinflammatory and immunoregulatory cytokines, to act as costimulatory cells, and to avoid spontaneous apoptosis in response to CD40L stimulation is markedly reduced.
The role of TNF-α in combating infections has recently been underscored by the finding that sepsis and other infectious complications developed in patients with rheumatoid arthritis who were treated with TNF-α antagonists (24
). Moreover, in clinical trials, immunotherapy against TNF-α significantly increased mortality (12
). Similarly, IL-1β plays an important role in the activation of innate, as well as adaptive, immunity. IL-1β induces neutrophil recruitment and plays a critical host defense role against Staphylococcus aureus
-caused brain abscesses, septic arthritis, and systemic infections (11
). In addition, this cytokine has been shown to influence the growth and differentiation of immunocompetent lymphocytes (30
). Finally, IL-12 is an immunoregulatory cytokine that is critical to the orchestration of cell-mediated immune responses in both the innate and adaptive immune system. IL-12 augments the production of IFN-γ and other cytokines from natural killer and T cells (6
). Furthermore, IL-12 appears to be a vital component of the host defense against gram-negative bacterial organisms, as evidenced by the heightened host resistance conferred by IL-12 administration in several bacterial infection models (18
). Therefore, the likely relevance of sepsis-related dysregulation of TNF-α, IL-1β, and IL-12 production to the increased risk of bacterial superinfection in survivors of sepsis is of considerable interest. On the other hand, it should be noted that mortality during sepsis may result from the development of multisystem organ failure, which is associated with the increased production of proinflammatory cytokines (4
). The production of proinflammatory cytokines in the early phases of sepsis is controlled in part by the innate immune response. However, CD40L-dependent or -independent monocyte CD40 activation has also been suggested to play a role. In particular, it has been reported that CD40 knockout mice had delayed death and improved survival after cecal ligation and puncture. The improvements in survival were associated with reduced serum levels of proinflammatory cytokines and IL-12 (15
). At variance with those results, we found that the CD40-mediated production of both proinflammatory cytokines and IL-12 is severely reduced in monocytes from septic patients from the early phases of the disease. This suggests a likelihood that the activation of monocytes by CD40 may not share a central role in the development of the early hyperinflammatory stages of sepsis.
In addition to reduced production of TNF-α, IL-1β, and IL-12, CD40L-activated monocytes from septic patients also showed a profoundly affected ability to upregulate CD80 and CD86 and to induce T-cell proliferation and cytokine secretion. A number of different cell types, including monocytes, perform antigen-presenting cell functions. However, to become competent antigen-presenting cells, monocytes first require activation by CD40L in order to upregulate the expression of CD80 and CD86 (53
). CD28/CD80-CD86 interactions play a central role in providing costimulatory signals to T cells (23
). The ligation of CD28 by CD80 and/or CD86 has been shown to induce T-cell production of growth factors and T-cell proliferation (8
). Thus, it is possible that the impaired costimulatory ability of CD40L-activated monocytes from septic patients was due to reduced surface expression of these molecules. In agreement with these findings, the results of recent studies indicate that the macrophage antigen-presenting capacity appears to become dysfunctional by 24 h after sepsis is induced (3
) and remains at subnormal levels for up to 14 days (14
). In contrast with the reduced ability to upregulate the expression of CD80 and CD86, CD40L-activated monocytes from septic patients proved able to optimally increase the expression of CD40. This indicates that the downmodulating effects of sepsis on CD40L-stimulated monocytes are not simply due to suboptimal CD40 expression. These data disagree with the results from our recent in vitro study in which LPS was found to interfere with CD40L-directed CD40 upregulation. The differences between the results of the in vitro and in vivo studies may be due to the presence in vivo of additional mediators able to affect the expression of coreceptors. Alternatively, it should be noted that in our in vitro experimental system the effect on CD40 expression was observed at LPS concentrations (>100 ng/ml) that are not usually detected during sepsis. In accord with the data presented here, several authors have reported that CD40 expression is unaltered or even enhanced on monocytes during sepsis (37
The results of recent studies of the means by which monocyte apoptosis is regulated have demonstrated that LPS activation or the treatment of monocytes with IL-1β or TNF-α protects monocytes from apoptosis (32
). Interestingly, cytokines involved in the recruitment of monocytes to sites of inflammation (e.g., monocyte chemoattractant protein 1 and transforming growth factor-β) have no such effect (31
). This suggests that monocytes may be recruited to a site of inflammation but will undergo apoptosis unless they receive further stimuli allowing for survival through activation. Signaling through CD40 has also been shown to regulate monocyte homeostasis during immune responses by counteracting apoptosis (41
). As we show here, monocytes from septic patients proved to be poorly responsive to the antiapoptotic effect of CD40 activation. The reduced ability of CD40L to rescue monocytes from apoptosis may increase the rate of monocyte apoptosis induced by antigen presentation (43
) and contribute to the decrease of the effectiveness of the immune response observed during sepsis.
Sepsis is a very complex syndrome due to the marked biologic differences of the etiologic determinants, heterogeneity of patients, and mostly unknown physiopathologic mechanisms. Thus, no single restricted patient population can be considered representative for this syndrome. Due to the very probable, literature-supported role of LPS in the pathogenesis of sepsis and, in particular, of monocyte dysfunction during sepsis, for this study we selected a cohort of patients with sepsis of documented gram-negative-organism etiology. With this background in mind, we wish to stress that any specific physiopathologic or therapeutic inference from our data should be made only with great caution at this time. However, a substantial share of the morbidity and mortality observed during sepsis is due to the consequences of sepsis-associated immunosuppression. Patients with severe sepsis may therefore benefit from treatments aimed at stimulating innate and adaptive immune responses. Monocyte hyporesponsiveness to CD40L may contribute substantially to the impairment of adaptive immune response during sepsis, and therapeutic strategies based on “boosting” the response of monocytes to CD40L may prove useful in ameliorating the clinical outcome in sepsis patients.