Saturday, 7 September 2013

The mighty neutrophil - not so mighty in ME/CFS

Neutrophils surrounded by erythrocytes (red blood cells)
Neutrophil granulocytes are the most abundant white blood cells in the body and represent a fundamental part of the innate immune system. Neutrophils are phagocytes which protect the body by engulfing and destroying microbes (typically bacterial and fungal pathogens). They are amongst the first immune cells attracted to areas of infection where they can migrate through blood vessel walls and infiltrate body tissue, a process guided by inflammatory signals (e.g. IL-8, IL-17 and leukotriene B4) and known as chemotaxis.

Neutrophils deal with microbes in several ways. Microbes can be internalised by phagocytosis and destroyed via oxidising radicals released during a respiratory burst. In addition neutrophils can release antimicrobials (e.g. oxidants and digestive enzymes) into the extracellular environment or generate neutrophil extracellular traps (NETs). During their activation release of various noxious and degrading substances ultimately kill and breakdown microbes. However these substances can also damage neutrophils themselves as well as surrounding tissue which may be of particular consequence in various chronic inflammatory diseases. As neutrophils become damaged and aged they themselves are phagocytosed by macrophages, which specialise in removing cellular debris.

Altered neutrophil function in CFS and its possible relation to immuno-pathology
In CFS several findings suggest neutrophil function is altered. A higher number of apoptotic neutrophils, lower numbers of viable neutrophils and increased expression of the death receptor TNFR1 was reported 1. Decreased neutrophil mitochondrial function has also been reported 2. Furthermore neutrophil respiratory burst capacity was found to be decreased while phagocytosis was unaffected 3. A recent study also reported that clinical response to the antiviral Valganciclovir (aka. Valcyte) was associated with an increase in neutrophil counts in CFS patients 4. However in contrast to these findings one study reported that basal neutrophil counts were elevated in CFS 5 although its use of the oxford diagnostic criteria challenges its validity. In my own personal case a persistently low neutrophil count is the only remarkable finding on typical blood counts.

So how does altered neutrophil function relate to other aspects of CFS immuno-pathology? Increased neutrophil apoptosis may be consistent with on-going infection since phagocytosis and sustained oxidative burst promote apoptosis 6–8. Accordingly raised levels of several immunological factors in CFS may serve to activate neutrophils (see table). For instance TNFα activates neutrophils via TNFR1/2-dependent activation of JNK and NF-KB 9. TNFα can also induce neutrophil apoptosis via TNFR1-dependent activation of a P38-PI3K pathway 9,10. This apoptosis is dependent upon oxidants generated via NADPH oxidase 10 and may be promoted during respiratory burst 8, which is discussed in more detail below.

The effects of selected agents on neutrophil function and survival
Bacterial components (e.g. LPS and peptidoglycan) Activate and prolong survival
TNF-α Activates and induces apoptosis
IL-1 Prolongs survival
IL-6 Activates
INF-γ Activates and prolongs survival
Inhibits function

Neutrophils also play important roles in the host defence against viral infections which themselves can modulate neutrophil activity in opposite directions. Increased neutrophil life span is induced by some viruses (e.g. RSV and CMV) and may contribute to chronic inflammation. Other viruses (e.g. SIV, HIV and influenza A) may increase neutrophil oxidative stress and apoptosis resulting in neutropenia and increased susceptibility to infection; this may also involve increased translocation of bacteria from the gut as a result of SIV/HIV enteropathology 11.

Altered neutrophil function may greatly impact the balance of the rest of the immune system in CFS. For instance apoptotic neutrophils normally promote the resolution of inflammation via interactions with macrophages. Initially apoptotic neutrophils signal to macrophages to ‘eat them’ and upon physical interaction change the transcriptional profile of the macrophage to release the immunosuppressive cytokines IL-10 and TGF-β 6. Consistent with this both IL-10 12,13 and particularly TGF-β 1,14–16 are increased in the periphery in CFS. However the simultaneous presence of other pro-inflammatory cytokines (e.g. TNFα) in CFS suggests on-going infection and immune activation incompletely inhibited by anti-inflammatory cytokines 13,17. Interestingly neutrophils were also recently demonstrated to be crucial to NK cell maturation and function 18, which may have relevance to the consistently decreased NK cell activity seen in CFS 3,12,13.

Neutrophil activity and redox in CFS
Further elucidation of neutrophil function in CFS may be achieved by considering redox (i.e. reduction-oxidation reactions, or in other words the balance between antioxidants and free radicals). At sites of inflammation large amounts of oxidants are generated by neutrophils and other immune cells (e.g. macrophages and eosinophils). For instance activation of neutrophils via various signaling pathways stimulates the respiratory burst 9 which involves the rapid uptake of oxygen and release of antimicrobial oxidants. The enzymes iNOS and NADPH oxidase produce nitric oxide (NO) and superoxide (O2-) respectively, and SODase further converts O2- to hydrogen peroxide (H2O2). The enzyme MPO then acts on H2O2 and chloride to produce the potent microbicidal oxidant hypochlorous acid (HOCl). (Note. some of these oxidants are used commercially as bleaches and disinfectants.)

Given the massive amounts of oxidants generated by phagocytes they are particularly dependent upon adequate antioxidants for protection; this is partly ensured by the use of reducing agents in the production of oxidants themselves. NAD(P)H is the major reducing molecule in cells and serves to maintain levels of reduced glutathione (GSH). NAD(P)H is utilised by both NADPH oxidase and iNOS to generate oxidants. Glutathione reduction is required to sustain phagocytic oxidative burst and promote the development of NETs 19. High levels of ascorbic acid (i.e. vitamin C) are required to prevent oxidation of the NOS cofactor BH4 which ultimately sustains neutrophil NOS expression, catalysis and oxidative burst 20. Taurine represents another antioxidant present at particularly high levels in phagocytes and is required for proper neutrophil activity and resolution of inflammation 21,22. During the respiratory burst taurine reacts with HOCl to form taurine chloramine which attenuates pro-inflammatory signaling and increases the expression of multiple antioxidant enzymes 21,22. The regulation of phagocyte function by redox extends to signaling molecules such as NF-KB 23. In short antioxidants are required for immune cells such as phagocytes to express their full potential.

The effects of selected agents on neutrophil respiratory burst
Glutathione Oxidative burst and NETs 19
Vitamin C BH4 stabilisation and NOS activity 20
Zinc Activates PKC-NADPH oxidase 24
Vitamin E
Inhibits PKC-NADPH oxidase 25
Calcium antagonism 26,27
Lowers radicals and glutathione 28

In CFS blood, general markers of cell-mediated immunity are elevated and relate to bacterial translocation from the gut and symptom severity 29,30. Upregulation of intracellular immuno-inflammatory NF-KB, COX and iNOS-NO pathways in peripheral lymphocytes has been reported 17. Markers of macrophage signaling are upregulated in the cerebrospinal fluid in CFS 31. Redox markers suggest oxidative stress occurs in blood, muscles and brain in CFS 17,32. Taken together these findings support the notion that general immune cell activation may be an important contributor to oxidative stress in CFS 17,32. Loss of redox may also impair the function of some immune cells themselves. Indeed a loss of antioxidants such as those discussed above impairs neutrophil phagocyte function 19–21, while in CFS blood measures of antioxidants (incl. glutathione 15, ascorbic acid 33, NAD(P)H 34 and taurine 35) are decreased. Furthermore a loss of redox balance promotes neutrophil apoptosis possibly via both intrinsic and extrinsic pathways 7. In CFS a loss of redox may therefore explain the reduced neutrophil burst capacity 3 and the increased proportion of apoptotic cells reported 1.

In summary, there has been very little research on neutrophils in CFS so far; however current findings seem consistent with the possibility that on-going infection drives phagocyte activation. Translocation of bacteria from the gut into peripheral blood may occur in a large subset of patients 17. In this case phagocyte activation may occur initially in the gut mucosa, while bacteria translocating into blood may further encounter kupffer cells, the resident macrophages of the liver, and systemic immune responses may also ensue 30. Chronic low-level activation and release of oxidants from immune cells could lead to exhaustion via a loss of intracellular redox which consequently limits optimal activity. This might contribute to the decreased neutrophil function in CFS and immunosuppression. Through changes in immuno-inflammatory signaling disturbed phagocyte function may further skew overall immune responses. While chronic low-level release of oxidants from struggling immune cells could generally promote systemic oxidative stress in CFS.

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