EFFECT OF LPS ON MICROGLIA

Shock 1998: Oxígeno, Oxido Nítrico y perspectivas terapéuticas
Simposio Internacional, Academia Nacional de Medicina
Buenos Aires, 30 abril 1998

Therapeutic implications of microglia activation by lipopolysaccharide and reactive oxygen species generation in septic shock and central nervous system pathologies: a review

Alejandro M.S. Mayer

Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, Illinois, USA

Key words: microglia, lipopolysaccharide, reactive oxygen species, superoxide, sepsis, shock, therapy, review.

Abstract

The pathophysiology of organ system failure in sepsis, in particular the effects of septic shock on the central nervous system, are still incompletely understood. Lipopolysaccharide(LPS) from Gram-negative bacteria affects the permeability of the blood-brain barrier and causes the activation of brain microglia. A growing body of research supports involvement of activated brain microglia in brain pathologies caused by infectious diseases, trauma, tumors, ischemia, Alzheimer’s disease, Parkinson’s disease, Down’s syndrome, multiple sclerosis and AIDS. Those seminal studies that have contributed to the characterization of the in vivo and in vitro effects of LPS on microglia function, mediator generation and receptor expression are presented within a historical perspective. In particular, all those in vitro studies on O2-, H2O2 and NO· generation by either unprimed or primed microglia have been extensively reviewed. The apparent controversial effect of LPS on microglia O2- is discussed. Because treatment modalities for septic shock have not significantly affected the current high mortality, alternative strategies with antioxidants are currently being investigated. Reduction of microglia O2- generation is proposed as a possible complementary strategy to antioxidative therapy for septic shock and CNS pathologies that involve activated microglia.

Resumen

Relevancia terapéutica de la activación de la microglia por lipopolisacárido y la generación de especies reactivas del oxígeno en el shock séptico y en patologías del sistema nervioso central: una revisión. En la actualidad no se hallan completamente establecidos los efectos del shock séptico sobre el sistema nervioso central(SNC). El lipopolisacárido(LPS) de bacterias Gram-negativas puede afectar la permeabilidad de la barrera hematoencefálica y causar la activación de la microglia en el SNC. Un creciente numero de investigaciones ha documentado el rol de la microglia activada en patologías del SNC causadas por diversos agentes infecciosos, trauma, tumores, isquemia, enfermedad de Alzheimer, síndrome de Down, esclerosis múltiple y síndrome de inmunodeficiencia adquirida. Se presenta una revisión de aquellos estudios, que desde una perspectiva histórica han contribuido a la caracterización de los efectos in vitro e in vivo del LPS sobre la activación de la microglia, la generación de mediadores y la expresión de receptores. En particular, se ha completado una detallada revisión de estudios in vitro sobre la generación de especies reactivas del oxígeno (ROS), en particular, O2-, H2O2 y NO· por parte de microglia activada o no activada. El aparente efecto contradictorio del LPS sobre la producción de O2- por parte de la microglia de rata ha sido comentado. Debido a que el tratamiento clínico actual del shock séptico no ha logrado disminuir la mortalidad de manera significativa, en la actualidad se investigan tratamientos alternativos. Un área de interés es el uso de antioxidantes para eliminar las ROS. Se propone que una alternativa al uso de antioxidantes es inhibir la generación del ROS por la microglia activada. Esta terapia alternativa podría afectar significativamente el tratamiento del shock séptico y de otras patologías del SNC.

Postal address: Dr. Alejandro M.S. Mayer, Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, Illinois 60515, USA. Fax: 630 - 971-6414; E-mail: amayer@midwestern.edu

Distributive shock, lipopolysaccharide and the septic mediator cascade

Shock is a complex clinical syndrome that regardless of etiology causes a profound reduction in tissue perfusion with inadequate delivery of oxygen to the brain and other vital organs1. Septic shock, a type of distributive shock characterized by massive arteriovenous dilation, is presently the leading cause of death in intensive care units and the thirteenth most common cause of death in the US2. Despite the importance to the outcome of septic shock, the pathophysiology of organ system failure and in particular the effects of sepsis on the central nervous system (CNS), one of the first organs to be affected by sepsis, are still incompletely understood3. Although septic shock may be caused by systemic infections with bacterial, fungal, mycobacterial, rickettsial, protozoal or viral organisms, the majority of clinical cases involve aerobic or anaerobic Gram-negative bacteremia. The latter are usually associated with nosocomial infections arising from urinary, gastrointestinal and respiratory tract infections by organisms like Escherichia coli, Klebsiella pneumoniae, Enterobacter-Serratia species, Proteus species and Pseudomonas aeruginosa4. Infections by Gram-negative bacteria like E. coli as well as Gram- positive organisms such as Streptococcus pneumoniae, cause inflammation in the subarachnoid space of the brain, the pathological hallmark of bacterial meningitis. This is a common neonatal worldwide disease that still has high mortality despite the availability of antimicrobial therapy5, 6. Gram-negative bacteria elicit systemic as well as CNS intrathecal inflammatory responses by releasing a structural component of their cell wall, namely lipopolysaccharide (LPS)7. As a bacterial factor, LPS was first isolated from Vibrio cholerae 106 years ago8. LPS can systemically activate endothelial cells, platelets, macrophage-monocytes and neutrophils to produce and release numerous endogenous mediators, including reactive oxygen intermediates (ROS) 9, 10, collectively known as the septic cascade. In the CNS, LPS- induced inflammation in the subarachnoid space leads to disruption of the blood-brain barrier11, 12, attraction of blood-derived leukocytes, release of inflammatory and neurotoxic mediators and activation of brain microglia5, 6, 13. Although a complete description of causal relationships between inflammatory mediators and clinical manifestations in septic shock remains to be fully elucidated, there is at present sufficient clinical and experimental evidence that septic inflammatory mediators, including ROS, are mainly responsible for the cardiovascular, pulmonary and CNS effects observed2.

The role of ROS in septic shock and other central nervous system pathologies.

In septic shock14 as well as in other CNS pathologies, such as Parkinson’s disease15, Alzheimer’s disease15, Down’s syndrome16, cerebral ischemia and reperfusion17, 18, amyotrophic lateral sclerosis19 and multiple sclerosis20 the generation of excessive quantitities of inflammatory cytokines13 such as, e.g. tumor necrosis factor-a21, interleukin-122, 23 as well as ROS17 and NO·24 has been well documented. There appear to be several potential mechanisms that could lead to the generation of ROS in the CNS : (1) mitochondrial electron transport chain electron bleed, (2) eicosanoid metabolism, (3) autooxidation of catecholamines, (4) xanthine oxidase and (5) the respiratory burst of activated leukocytes14, 17, 18. Generation of ROS by the first four mechanisms has received intense scrutiny by investigators over the past 20 years, with the use of a wide variety of techniques14. But generation of ROS by CNS leukocytes, i.e. infiltrating neutrophils and monocytes as well as resident microglia production of superoxide anion (O2-), hydrogen peroxide (H2O2) and nitric oxide (NO·) in the CNS, has only received notable attention since the mid-1980’s25-27 (Table 1 and 2). The underlying pathophysiological effects of ROS in these CNS pathologies, in particular those related to the production of the radical O2- and the non-radical H2O2, have been shown to result from DNA strand breaks, protein alterations and the formation of lipid hydroperoxides which may disrupt membrane function as well as membrane-bound enzymes and receptors14, 17, 18, 20. Because the brain is rich in iron content28 ROS injury to brain cells could potentially result in iron ion release, further free radical formation and damage to neurons, particularly their synapses29 as well as to oligodendrocytes, the myelin producing cell of the central nervous system30. Interestingly ROS do not seem to affect microglia or astrocytes31. Prolonged exposure to ROS could override normal CNS antioxidant defense mechanisms, e.g. superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, permanently affecting cellular function32. Finally, although excessive production of ROS can lead to CNS pathology, ROS do fulfill physiological functions in the brain where they appear to be involved in intracellular signalling33 as well as normal CNS metabolism15, 34.

Microglia activation by LPS and the generation of ROS

It has been known for more than 25 years35 that activated phagocytes such as monocytes, tissue macropha-ges, neutrophils and eosinophils are able to generate ROS. Since 1986, numerous studies have shown that the leukocyte-dependent source of O2- in the CNS is the microglia, the brain resident macrophage (Table 2). Microglia are leukocytes derived from outside the CNS, and as first proposed by Del Rio Hortega36 in 1932, they are formed in the bone marrow, then enter the circulation as monocytes and migrate into the brain during late embryonic life establishing permanent residency37. Histological studies have shown that in the normal brain microglia are of two types, ameboid microglia found in perinatal CNS and ramified microglia found throughout the gray and white parenchyma of the adult CNS. The historical controversies regarding the origin of the microglia have been extensively reviewed37, 38, 39 . Microglia activation in brain pathologies, as caused by infectious diseases, inflammation, trauma, brain tumors, ischemia and AIDS, may result in neuronal injury and ultimately neurodegeneration40, 41, 42, 43, 44. Similar to other tissue macrophages, when activated microglia release a large number of secretory products35, 45, 46, followed by sublethal and lethal injury to the CNS. Two different phenotypic forms of microglia appear, the activated but nonphago-cytic microglia in inflammatory pathologies and the reactive or phagocytic microglia in trauma, infection and neuronal degeneration. Both appear to have the capacity to express cell-surface receptors and release biologically active substances known to be mediators of inflammation, such as cytokines, coagulation factors, complement factors, eicosanoids, proteases, ROS and NO· 44,47.
One of the activators of microglia ROS generation that has received growing attention over the past 5 years is LPS9. LPS potently activates macrophages mediator generation via the lipid A portion of the macromolecule48. Similar to Kupffer cells49,50, unstimulated parenchymal brain microglia appear to be downregulated in terms of endocytic, cell surface receptor expression 51 and ROS generation52. However, LPS as well as the mediators elicited by LPS in septic shock, have been shown to affect permeability of the brain microvasculature 6,12,22,53, and to induce activation of brain microglia in vivo 54. The publications listed in Table 1 document in historical order the earliest published reports of in vivo53-60 and in vitro 60-93 effects of LPS on the activation of microglia effector functions. These functions include cytotoxicity61,81,90, antigen expression66,78,91,92, growth inhibition62, ion channels74, cytoskeletal changes72, 77, bacterial digestion88 and possibly apoptosis93. Furthermore, once activated microglia generate a vast array of mediators that include growth factors63, 87, ROS76, NO·85, 87, 97, complement70, 87, proteases67, 79, 84, excitatory aminoacids75, arachidonic acid derivatives62, 82 and cytokines60, 64, 68, 71, 73, 80, 86, 89, possibly by affecting microglia signal transduction mechanisms83. A similar historical perspective was used to prepare Table 2 which clearly demonstrates that since 198625, when microglia were first shown to have the same capacity as other macrophages to generate O2-, there has been a great interest in characterizing the mechanism of ROS generation by this brain phagocyte. During the past 12 years, numerous research groups have shown that O2-, H2O2 and NO· are generated by microglia isolated from rats 25, 26, 52, 65, 95, 97, 100-102, 104-106, 109, 111-113, 115, 116, 118, 120, 121, mi-ce27, 76, 98, 99, 103, 114, 117, hamsters114, dogs96, swine108 and humans107, 108, 110, 114, 119, when stimulated with a variety of agonists such as phorbol ester25-27, 52, 76, 95, 98, 100, 102, 104,106-108, 113, 115, 116, 118, opsonized zymosan26, 27, 100, 103, 114, 118, calcium ionophore105, antiviral antibodies96, antibody-coated red blood cells27, LDL111 and myelin112. Furthermore, these studies have clearly demonstrated that unprimed25-27, 96, 98, 102, 104, 105, 111, 112 microglia in vitro ROS generation is enhanced when these phagocytes are primed with interferon a, b and g.95, 99, 100, 107, 108, 110, 119, TNF-a107,108,110,114, interleukin-1110, 114, 119 , b-amyloid 109, 113, 118, albumin115 and LPS52, 65, 76, 101, 103, 106, 108, 113, 114, 116, 117, 120, 121 prior to agonist stimulation .
Interestingly, the effect of LPS on in vitro generation of agonist-stimulated microglia O2- generation appears to be controversial. While Colton et al.106 reported that « LPS does not affect O2- production in rat microglia «, other investigators have reported that LPS primes mouse76 and rat113 microglia for enhanced PMA-stimulated O2- release. Our recent report confirms these two studies52. Furthermore, we have observed that LPS concentrations greater than 3 ng/ml will exert a cytotoxic effect on neonatal microglia52 in vitro. Thus far, our experimental results suggest that LPS has a biphasic in vitro effect on microglia O2- generation: at concentrations lower than 3 ng/ml, LPS potently and dose-dependently primes PMA-stimulated O2- generation; however, at concentrations greater than 3ng/ml, LPS appears to inhibit PMA-stimulated O2- generation. Concomitant with this inhibition of O2- generation, we have measured enhanced release of NO·, TNF-a, thromboxane B2, metalloproteinases MMP-9 and LDH, as well as apoptosis93. Since differences in O2- generation have been reported in human, mice and hamster microglia114, determining if LPS’s biphasic effects on rat microglia will also occur in microglia of other species, particularly in human microglia, appears to be an important question that needs to be answered. Systemic administration of LPS activates rat microglial cells54 in the hypothalamus, thalamus and brainstem. In human pathologies like septic shock and bacterial meningitis, if microglia, once activated release mediators such as TNF-a, potential toxicity could result to neurons, oligodendrocytes and astrocytes. Finally, determining if microglia ROS generation will be activated in experimental sepsis with the more virulent strains of E. coli, serotypes that can survive and disseminate outside the intestine and ultimately cause human septicemia, needs to be addressed2, 122. Interestingly, none of the publications listed in Tables 1 or 2 used E. coli LPS derived from the virulent strains2, 122.

Alternative therapeutic strategies for septic shock and the modulation of microglia ROS generation

Since certain areas of the brain are rich in the transition metal iron and microglia O2- has been shown to release iron from ferritin94, H2O2 may combine with Fe2+ ions to form the highly reactive ·OH123, which can then initiate multiple cellular lesions124,20. Microglia have also been shown to release NO·65, which can react with O2- to form peroxynitrite125 at a rate constant that is three times faster than the rate at which superoxide dismutase scavenges O2-. Peroxynitrite is a powerful oxidant that has been shown to oxidize sulfhydryl groups, lipids, DNA and proteins125. However, O2-, H2O2 and ·OH normally have potent bactericidal functions, and thus the generation of microglia ROS under physiological conditions, when NO· concentration is 100-fold lower than that of superoxide dismutase, could serve to protect the CNS against infectious organisms, ie. bacteria, fungi and viruses 33,125,126.
Current treatment for septic shock includes antimicrobial chemotherapy, radiologic and surgical procedures, volume replacement, inotropic and vasoconstrictor support, oxygen therapy , mechanical ventilation as well as hemodialysis and hemofiltration1 . However, these treatments appear to have been unsuccessful in diminishing the high mortality associated with septic shock2,127, 128. Since 1987, 12 prospective, placebo-controlled, rando-mized, double-blind, multicenter trials involving a total of 6266 patients with gram-negative sepsis have failed to demonstrate clinical efficacy of these and other treatment strategies including methylprednisolone, anti-LPS antibodies, platelet-activating factor-receptor antagonist, recombinant human IL-1 receptor antagonist, anti-TNF antibodies and ibuprofen2. Another strategy being investigated for septic shock is antioxidative therapy129, which is also being extensively studied for treatment of neurological infections130 and other CNS pathologies131 where ROS have been implicated. However, if as discussed earlier under pathological conditions like septic shock, NO· is produced in sufficient quantities so that it can physiologically outcompete superoxide dismutase for O2-, production of significant amounts of peroxynitrite125 would occur, as opposed to dismutation of O2- to H2O2. It would therefore seem reasonable to conclude that enhanced microglia O2- generation in brain pathologies will be only partially modulated by the use of antioxidants that scavenge ROS. Alternative pharmacological strategies, specifically targeted to turn off or reduce rather than scavenge microglia O2-, by targeting signal transduction pathways leading to NADPH oxidase activation, might be a better approach that may hold considerable clinical promise. Why modulate micro- glia generation of O2- as opposed to simply scavenging ROS ? As explained earlier, modulation of O2- may help preserve physiological levels of ROS which are necessary for the role of the microglia in the defense of the brain126 and antioxidants such as SOD may be unable to prevent rapid formation of peroxynitrite in brain pathologies where NO· production is elevated thus fail to protect against damage to neurons and other cells in the brain125. The reduction of O2- generation in LPS-activated leukocytes by targeting signal transduction pathways leading to O2- generation has been studied in rat PMN132, rat Kupffer cells50, rat alveolar macrophages133 and recently in retinoic acid-activated human promyelocytic leukemia cells134. Although scavenging ROS is certainly a potential treatment for activated microglia135, reducing the generation of NADPH-dependent respiratory burst oxidase-dependent ROS134 might be a particularly useful approach in those conditions where microglia are primed for enhanced O2- generation136. Hopefully, in the future the combination of these two approaches will contribute to the successful development of alternative therapies for septic shock and brain pathologies that involve activated microglia and the generation of ROS.

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Table 1: Effects of LPS on microglia function, mediator generation and receptor expression:
a historical perspective

Year Species LPS Study completed Effect on Reference
In vitro In vivo

1986 Rat E. coli (NR) x IL-1 Giulian60
1987 Mice E. coli (NR) x Tumor cytotoxicity Frei61
1989 Rat S. typhimurium x Growth Gebicke62
1989 Rat S. typhimurium x PGE2 Gebicke62
1989 Rat NR x NGF Mallat63
1989 Mouse NR x TNF-a Sawada64
1992 Mice S. abortus equi x Activation Andersson55
1992 Rat NR x NO· Boje65
1992 Mice NR x CD4 receptor Sawada66
1992 Rat E. coli (NR) x Elastase Nakajima67
1992 Rat NR x IL-1 Yao68
1992 Rat E. coli 055 B5 x IL-1 Van Dam56
1993 Mice E. coli 0111:B4 x Toxoplasma gondii Chao69
1993 Mice E. coli 0111:B4 x C3 Haga70
1993 Human E. coli 055:B5 x IL-1; IL-6, TNF-a Lee71
1994 Rat NR x Ca2+/Actin Bader72
1994 Mice NR x MSR Receptor Bell57
1994 Mice NR x IL-10 Mizuno73
1994 Rat S. typhimurium x K+ channels Norenberg74
1994 Rat/mouse E. coli 026:B6 x Glutamate Patrizio75
1994 Rat E. coli 055:B5 x MHC I/II Antigen Xu58
1994 Mouse E. coli 0111B4 x O2- generation Chao76
1995 Mouse E. coli 0127:B8 x Cytoskeleton Basset77
1995 Rat E. coli 055 B5 x IL-3 receptor Appel78
1995 Mouse S. abortus equi x ICAM-1 Bell59
1995 Rat E. coli 055:B5 x Gelatinases Gottschall79
1995 Mouse NR x b-chemokines Hayashi80
1995 Rat S. pneumoniae x Toxicity Kim81
1995 Rat E. coli 026:B6 x PGD2,TXB2 Minghetti82
1995 Rat E. coli 026:B6 x cAMP Patrizio83
1995 Mouse E. coli 0127:B8 x Cathepsin B Ryan84
1995 Rat E. coli 055:B5 x Biopterin Sakai85
1995 Mice E. coli (NR) x IL-2 Sawada86
1995 Human E. coli 055:B5 x C1q/C3; TGFb; NOS Walker87
1996 Rat E. coli 055 B5 x CD11b/c & MHC II Buttini54
1996 Mouse S. aureus x Bacterial digestion Fincher88
1996 Mouse NR x IL-12 Lodge89
1996 Mouse NR x Neurons Zhang90
1997 Mouse E. coli 026:B6 x B7 antigen Iglesias91
1998 Rat E. coli 055:B5 x CD54/CD29 Zuckerman92
1998 Rat H. influenzae BBB Permeability Wispelwey53
1998 Rat E. coli 026:B6 x Apoptosis Mayer93

Abbreviations: BBB: blood brain barrier; C: complement; FGF: fibroblast growth factor; IL: interleukin; MSR: macrophage scavenger receptor; NOS: nitric oxide synthase; NGF: nerve growth factor; NR: bacterial species or LPS serotype not specified; TGF: transforming growth factor;
Table 2: In vitro studies on the mechanism of microglia ROS production: a historical perspective.

Year Microglia Priming agent Agonist ROS Reference
source studied

1986 Rat - PMA O2- Giulian25
1987 Rat - TPA/OZ O2- Colton26
1987 Mouse - TPA/OZ/IgRB O2- Sonderer27
1989 Rat IFN g PMA O2- (+) Woodroofe95
1989 Dog - Antiviral Ab O2- Burge96
1991 Mouse LPS/IFN-g/GM-CSF - O2- Suzumura97
1992 Rat LPS - NO· Boje65
1992 Mouse - PMA O2-,H2O2 Piani98
1992 Mouse IFN-h LPS NO· Chao99
1992 Rat INF a,b,g PMA/OZ O2- (+) Colton100
1992 Rat LPS/IFN-g - NO· Zielasek101
1993 Rat - PMA H2O2 Banati102
1993 Mouse LPS OPZ NO· Corradin103
1994 Rat - PMA/ConA/OZ O2- Klegeris104
1994 Rat - A23187 O2- Colton105
1994 Rat LPS PMA O2- (-) Colton106
1994 Mouse LPS PMA O2- (+) Chao76
1995 Human TNF-a/IFN-g PMA O2- (+) Chao107
1996 Human TNF-a PMA O2- (+) Hu108
1996 Swine IFN-h+ LPS - NO· Hu108
1996 Rat b-amyloid - NO· Ii109
1996 Human IL-1/TNF-a/IFN-g - O2-/NO·(+) Janabi110
1996 Rat - LDL NO· Mohan111
1996 Rat - Myelin O2-/NO· Mosley112
1996 Rat b amyloid PMA O2- (+) Van Muiswinkel113
1996 Rat LPS PMA O2- (+) Van Muiswinkel113
1996 Mouse/Hamster LPS OZ O2-/NO·(-+) Colton114
/Human IL-1 + TNF-a
1997 Rat LPS PMA O2- (+) Mayer52
1997 Rat Albumin PMA O2- (+) Si115
1997 Rat LPS PMA O2-/NO· (-) Si116
1997 Mouse LPS - NO· Murata117
1997 Rat b amyloid OZ/PMA O2- (+) Klegeris118
1997 Human INF-g/IL-1 - NO· Ding119
1998 Rat LPS - NO· Bhat120
1998 Rat LPS - NO· Lockhart121

Abbreviations: Ab: antibody; A23187: calcium ionophore A23187; ConA: concanavalin A; IFN; interferon; IgRB: antibody-coated red blood cells; LDL: low density lipoprotein; OZ: opsonized zymosan; PMA: phorbol myristate acetate; (+) increased production; (-) decreased production