The present study demonstrates that neutrophils have an accelerated rate of apoptosis in patients with infection. E. coli did not promote neutrophil apoptosis directly; nevertheless, E. coli and S. aureus enhanced monocyte expression of FasL. Moreover, sera (irrespective of coming from gram-positive or gram-negative patients) promoted neutrophil apoptosis, indicating the presence of a circulating factor in sera. Interestingly, anti-FasL antibody attenuated infected-serum-induced neutrophil apoptosis. Infected patients also showed an increased content in serum of FasL. Conditioned media from activated macrophages derived from FasL-deficient mice induced less apoptosis in human and murine neutrophils than conditioned media of control macrophages. These results suggest that infection enhances neutrophil apoptosis through FasL which may have been released in the sera from microorganism-activated monocytes.
Neutrophils are the most abundant of all leukocytes. The life of a neutrophil is spent in three environments: marrow, blood, and tissues. Proliferation and maturation take place in the marrow over a period of 13 to 15 days (32
), after which mature neutrophils enter the blood stream. This period can be shortened during times of stress. Under steady-state conditions, death and clearance in the tissues balance daily production of neutrophils. Neutrophils are programmed to die by apoptosis at the time of differentiation. Although inevitable, the exact timing of neutrophil death is subject to regulation by external factors (4
Host defense in response to bacterial invasion utilizes both the innate and acquired immune response. Neutrophils appear to be the important mediators of innate immunity. This is exemplified by the occurrence of recurrent infections in patients with neutropenia (38
). Moreover, the susceptibility of humans with neutrophil dysfunction to a wide array of microbial infections would also appear to be good evidence of the importance of neutrophils in microbial clearance in the early stage of infection. With this background, it appears that increased apoptosis of the neutrophils during infection may be an important step in the transition of the host response from its initial stage (which relies predominantly on the components of the innate response, such as neutrophils) to its intermediate and later stages (which rely more heavily on the components of acquired immunity, such as monocytes and antibodies). The present study suggests that neutrophilia may not always mean increased numbers of functioning neutrophils. Moreover, the promotion of apoptosis by infection may transfer host dependency from an innate response to an acquired immunity.
In vivo, apoptotic neutrophils are rapidly cleared by neighboring phagocytes (3
). This mechanism of cell death and clearance of neutrophils has been postulated to represent an injury-limiting process (3
). Death by necrosis would result in widespread release of neutrophil cytoplasmic enzymes and subsequent extensive tissue destruction. Apoptosis is a highly regulated and genetically directed process. It is induced by intracellular cues, such as DNA damage or osmotic stress, and extracellular cues, including growth factor withdrawal, matrix detachment, and direct cytokine-mediated killing (15
). Two main pathways, caspase proteases or the mitochondrial pathway, are involved in apoptotic cell death (15
). However, at every level the action of proapoptotic molecules is opposed by a set of inhibitors. A number of signals such as interaction of TNF-FasL with its respective cognate receptor, TNF-R–Fas, induce trimerization of the receptors (TNF-R1, Fas, DR3, DR4, DR5, and DR6 all contain an intracellular “death domain”), recruiting adapter proteins such as FADD to the death domain (17
). These adapter molecules then recruit and activate caspase 8. In the mitochondrial pathway, in response to apoptotic signals, proapoptotic bcl-2 family members translocate to and alter the permeability of the mitochondrial membrane, cytochrome c
release, and the production of reactive oxygen species. Antiapoptotic members of the bcl-2 family, such as bcl-2, reside in the mitochondrial membrane and may counter these effects. Both caspase and mitochondrial pathways are interconnected; i.e., caspase 8 cleaves Bid (of the bcl-2 family) to produce tBid; in turn, tBid in cooperation with Bad (of the Bcl-2 family) can trigger cytochrome c
release, inducing the caspase adapter Apaf-1 to activate caspases 9 and 3. Finally, activation of caspase 3 either through cleavage of caspase 8 or through the mitochondrial pathway causes degradation of proteins. In the present study, Fas (APO-1; CD95)-FasL interaction has triggered apoptosis of neutrophils. We believe that the downstream signaling of infection-induced neutrophil apoptosis involves recruitment of the adapter protein, FADD, followed by caspase 8 and caspase 3 activation. Since phagocytization of microorganisms by neutrophils is associated with the generation of reactive oxygen species, the mitochondrial pathway also seems to be operative. Whether Fas-FasL interaction and generation of reactive oxygen species are interrelated and/or have additive or synergistic effects on neutrophil apoptosis requires future studies. We plan to probe infection-induced neutrophil downstream signaling in future studies.
Human immunodeficiency virus type 1 gp120 envelope protein has been shown to increase FasL expression in monocytes and to enhance the expression of FasL gene transcription via the FasL gene enhancer-promoter region upregulation (26
). Upregulation of FasL mRNA and the associated increase in apoptosis of Fas-susceptible targets have been reported previously (2
). Interestingly, macrophage-associated FasL has been shown to trigger selective apoptosis of Fas-susceptible CD4 but not CD8 T cells from human immunodeficiency virus-positive patients (1
Recently, Brown and Savill demonstrated that exposure of monocytes/macrophages to opsonized zymosan induced the release of soluble FasL (3
). The conditioned supernatants containing FasL led to Fas-mediated apoptosis of bystander monocytes and FasL-negative neutrophils. Although macrophages phagocytizing latex beads produced soluble FasL, it did not show proapoptotic effects on neutrophils (3
). For the occurrence of bystander cell apoptosis, these investigators hypothesized a requirement of additional soluble factors besides soluble FasL (3
Recently it was reported that E. coli
directly triggers macrophage apoptosis (33
). To determine the molecular mechanism of E. coli
-induced macrophage apoptosis, in the present study we evaluated the effect of E. coli
on monocyte mRNA expression of FasL. Interestingly, E. coli
promoted monocyte mRNA expression of FasL. Since activated monocytes have been demonstrated to express Fas, we propose that E. coli
-induced monocyte FasL expression and subsequent Fas-FasL cross-linking may trigger autocrine cell death. Neutrophils have also been demonstrated to express Fas. Interaction between infection-induced increase of FasL (released from circulating monocytes) in serum and neutrophil Fas receptors may accelerate neutrophil apoptosis. In the present study, bacteria did not promote apoptosis of neutrophils; however, sera of infected patients enhanced neutrophil apoptosis, indicating the presence of a soluble factor. That factor seems to be FasL.
We conclude that patients with infection show accelerated neutrophil apotosis. This effect of infection on neutrophils seems to be mediated through FasL. We propose that microorganisms may be compromising the innate response and may thus make the host more dependent on acquired immunity. Since microorganisms are capable of promoting apoptosis in neutrophils and monocytes, a transfer of the host defense burden from innate to acquired immunity may be a forced rather than a sound strategy on the part of the host defense system and thus may not always be associated with a positive outcome. The present study provides a plausible explanation for the lack of correlation between neutrophilia and the outcome of infection.