MEDICINA - Volumen 58 - N°4, 1998
MEDICINA (Buenos Aires) 1998; 58: 377-385




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.


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.


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:


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.


1. Arrigoni LE, Rogers DK. Intensive Care Therapeutics. In: Young LY, Koda-Kimble MA, (eds). Applied Therapeutics: The clinical use of drugs. Vancouver:Applied Therapeutics, Inc., 1995; 1802-35.
2. Deitch EA. Animal models of sepsis and shock: a review and lessons learned. Shock 1998: 1:1-11.
3. Bowton DL. CNS effects of sepsis. Crit Care Clin 1989; 5: 785-92.
4. Weinstein MP, Towns ML, Quartey SM, et al. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in Adults. Clinic Infec Dis 1997; 24: 584-602.
5. Lavoie FW. Meningitis, encephalitis, and central nervous system abscess. In: Rosen P, Barkin R. (eds). Emergency Medicine. Concepts and Clinical Practice. St. Louis: Mosby-Year Book Inc., 1998; 2198-2211.
6. Tunkel AR, Scheld WM. Pathogenesis and pathophy- siology of bacterial meningitis. Annu Rev Med 1993; 44: 103-20.
7. Holst O, Ulmer AJ, Brade H, Flad HD, Rietschel ET. Biochemistry and cell biology of bacterial endotoxins. Minireview. FEMS Inmunol Med Microbiol 1996; 16: 83-104.
8. Pfeiffer R. Untersuchungen uber das Choleragift. Z Hyg Infektionskr 1892; 11: 393-412
9. Burrell R. Human responses to bacterial endotoxin. Circ Shock 1994; 43: 137-53.
10. Wright SD. Multiple receptors for endotoxin. Curr Opin Immunol 1991; 3: 83-90.
11. Pfister HW, Scheld WM. Brain injury in bacterial meningitis: therapeutic implications. Curr Opin Neurol 1997; 10: 254-59.
12. Abbott NJ, Revest PA. Control of brain endothelial permeability. Cerebrovasc Brain Metab Rev 1991; 3: 39-72.
13. Benveniste EN. Inflammatory cytokines within the central nervous system: sources, function and mechanism of action. Am J Physiol 1992; 263: C1-C16.
14. Zimmerman JJ. Oxygen free radicals. In: Chernow B (ed). The Pharmacologic approach to the critically ill patient. Williams & Wilkins, 1994; 901-25.
15. Halliwell B. Reactive oxygen species and the central nervous system. J Neurochem 1992; 59: 1609-23.
16. Colton CA, Yao J, Gilbert D, Oster-Granite ML. Enhanced production of superoxide anion by microglia from trisomy 16 mice. Brain Res 1990; 519: 236-42.
17. Chan PH. Role of oxidants in ischemic brain damage. Stroke 1996; 27: 1125-9.
18. Kontos HA. Oxygen radicals in CNS damage. Review Article. Chem Biol Interact 1989; 72: 229-55.
19. Bergeron C, Muntasser S, Somerville MJ, Weyer L, Percy ME. Copper/zinc superoxide dismutase mRNA levels are increased in sporadic amyotrophic lateral sclerosis motorneurons. Brain Res 1994; 659: 272-6.
20. Ruuls SR, Bauer J, Sontrop K, Huitinga I, Hart BA, Dijkstra CD. Reactive oxygen species are involved in the patho-genesis of experimental allergic encephalomyelitis in Lewis rats. J Neuroimmunol 1995; 56: 207-17.
21. Deli MA, Descamps L, Dehouck MP, et al. Exposure of TNF-a to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase in permeability and cytoplasmatic stress fiber formation of actin. J Neurosci Res 1995; 41: 717-26.
22. Quagliarello VJ, Wispelwey B, Long WJ, Scheld WM. Recombinant human interleukin-1 induces meningitis and blood-brain barrier injury in the rat. J Clin Invest 1991; 87: 1360-6.
23. Rothwell NJ. Functions and mechanisms of interleukin 1 in the brain. Trends Pharmacol Sci 1991; 12: 430-6.
24. Dawson VL, Dawson TM. Nitric oxide in neuronal dege-neration. Proc Soc Exp Biol Med 1996; 211: 33-40.
25. Giulian D, Baker TJ. Characterization of ameboid microglia isolated from developing mammalian brain. J Neurosci 1986; 6: 2163-78.
26. Colton CA, Gilbert DA. Production of superoxide anions by a CNS macrophage, the microglia. FEBS Lett 1987; 223: 284-8.
27. Sonderer P, Wils P, Wyler R, Fontana A, Peterhans E, Schwyzer M. Murine glial cells in culture can be stimulated to generate reactive oxygen. J Leukoc Biol 1987; 42: 463-73.
28. Dwork AJ, Schon EA, Herbert J. Nonidentical distribution of transferrin and ferric iron in human brain. Neuroscience 1988; 27: 333-45.
29. Colton CA, Fagni L, Gilbert D. The action of an oxygen intermediate, H2O2, on synaptic transmission produced by hydrogen peroxide. J Free Rad Biol Med 1989; 7: 3-8.
30. Szuchet S, Polak P, Yim, SH. Mature oligodendrocytes cultured in the absence of neurons recapitulate the ontogenic development of myelin membranes. Dev Neurosci 1986; 8: 208-21.
31. Griot C, Vandevelde M, Richard A, Peterhans E, Stocker R. Selective degeneration of oligodendrocytes mediated by reactive oxygen species. Free Radic Res Commun 1990; 11: 181-93.
32. Fridovich I. Superoxide dismutase. Annu Rev Pharmacol Toxicol 1983; 23: 239-57.
33. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide; phy-siology, pathophysiology and pharmacology. Pharmacol Rev 1991; 43: 109-42.
34. Byczkowski JZ, Gessner T. Biological role of superoxide ion-radical. Minireview. Int J Biochem 1988; 20: 569-80.
35. Nathan CF. Secretory products of macrophages. J Clin Invest 1987; 79: 319-26.
36. Del Rio Hortega P. In Cytology and Cellular Pathology of the Nervous System. 1932; (Penfield, W. ed.) 482-534, Paul Hoeber, New York.
37. Lassman H, Schmeid M, Vass K, Hickey WF. Bone marrow derived elements and resident microglia in brain inflammation. Glia 1993; 7: 19-24.
38. Ling EA, Wong WC. The origin and nature of ramified and amoeboid microglia: a historical review and current concepts. Glia 1993; 7: 9-18.
39. Theele DP, Streit WJ. A chronicle of microglial ontogeny. Glia 1993; 7: 5-8.
40. Banati RB, Gehrmann J, Schubert P, Kreutzberg GW. Cytotoxicity of microglia. Glia 1993; 7: 111-8.
41. Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends Neurosci 1996; 19: 312-318.
42. Dickson DW, Lee SC, Mattiace LA, Yen SH, Brosnan C. Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer’s disease. Glia 1993; 7: 75-83.
43. Lassmann H, Hickey WF. Dynamics of microglia in brain pathology. Clin Neuropathol 1993; 12: 284-5.
44. Gehrmann J, Matsumoto Y, Kreutzberg GW: Microglia: intrinsic immunoeffector cell of the brain. Brain Res Rev 1995; 20: 269-87.
45. Johnston RB. Monocytes and macrophages. N Engl J Med 1988; 318: 747-52.
46. Laskin DL, Pendino KJ: Macrophages and inflammatory mediators in tissue injury. Annu Rev Pharmacol Toxicol 1995; 35: 655-677.
47. McGeer PL, McGeer EG. The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res Rev 1995; 21: 195-218.
48. Doe WF, Yang ST, Morrison DC, Betz SJ, Henson PM. Macrophage stimulation by bacterial lipopolysaccharides. II. Evidence for differentiation signals delivered by lipid A and by a protein rich fraction of lipopolysaccharides. J Exp Med 1978; 148: 557-68.
49. Mayer AMS, Spitzer JA. Continuous infusion of Escheri-chia coli endotoxin in vivo primes in vitro superoxide anion release in rat polymorphonuclear leukocytes and Kupffer cells in a time-dependent manner. Infect Immun 1991; 59: 4590-8.
50. Mayer AMS, Spitzer JA. Modulation of superoxide generation in in vivo lipopolysaccharide-primed Kupffer cells by staurosporine, okadaic acid, manoalide, arachidonic acid, genistein and sodium orthovanadate. J Pharmacol Exp Ther 1994; 268: 238-247.
51. Perry VH, Gordon S. Macrophages and the nervous system. Int Rev Cytol 1991; 125: 203-44.
52. Mayer AMS, Oh S, Presto E, Glaser KB, Jacobson P. LPS-primed rat brain microglia: a convenient in vitro model to search for antiinflammatory marine natural products. Shock 1997; 7(S2): 49.
53. Wispelwey B, Lesse AJ, Hansen EJ, Scheld WM. Haemo-philus influenzae lipopolysaccharide-induced blood brain barrier permeability during experimental meningitis in the rat. J Clin Invest 1998; 82: 1339-46.
54. Buttini M, Limonta S, Boddeke HW. Peripheral adminis-tration of lipopolysaccharide induces activation of microglial cells in rat brain. Neurochem Int 1996; 29: 25-35.
55. Andersson PB, Perry VH, Gordon S. The acute inflamma-tory response to lipopolysaccharide in CNS parenchyma differs from that in other body tissues. Neuroscience 1992; 48: 169-86.
56. Van Dam, AM, Brouns M, Louisse S, Berkenbosch. Appearance of interleukin-1 in macrophages and in ramified microglia in the brain of endotoxin-treated rats: a pathway for the induction of non-specific symptoms of sickness. Brain Res 1992; 588: 291-6.
57. Bell MD, Lopez-Gonzalez R, Lawson L, et al. Upregulation of the macrophage scavenger receptor in response to different forms of injury in the CNS. J Neurocytol 1994; 23: 605-13.
58. Xu J, Ling EA. Upregulation and induction of surface antigens with special reference to MHC class II ex-pressions in miroglia in postnatal rat brain following intravenous or intraperitoneal injections of lipopolysaccha-ride. J Anat 1994; 184: 285-96.
59. Bell MD, Perry VH. Adhesion molecule expression on murine cerebral endothelium following the injection of a proinflammagen or during acute neuronal degeneration. J Neurocytol 1995; 24: 695-710.
60. Giulian D, Baker TJ, Shih LN, Lachman LB. Interleukin-1 of the central nervous system is produced by ameboid microglia. J Exp Med 1986; 164: 594-604.
61. Frei K, Siepl C, Groscurth P, Bodmer S, Schwerdel C, Fontana A. Antigen presentation and tumor cytotoxicity by interferon g-treated-microglial cells. Eur J Immunol 1987; 17: 1271-8.
62. Gebicke-Haerter PJ, Bauer J, Schobert A, Northoff H. Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells. J Neurosci 1989; 9: 183-94.
63. Mallat M, Houlgatte R, Brachet P, Prochianz A. Lipopoly-saccharide-stimulated rat brain macrophages release NGF in vitro. Dev Biol 1989; 133: 309-11.
64. Sawada M, Kondo N, Suzumura A, Marunouchi T. Produc-tion of TNF-a by microglia and astrocytes in culture. Brain Res 1989;401: 394-7.
65. Boje KM, Arora PK. Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res 1992; 587: 250-6.
66. Sawada M, Suzumura A, Marunouchi T. Down regulation of CD4 expression in cultured microglia by immuno-suppressants and lipopolysaccharide. Biochem Biophys Res Commun 1992; 189: 869-76.
67. Nakajima K, Shimojo M, Hamanoue M, Ishiura S, Sugita H, Kohsaka S. Identification of elastase as a secretory protease from cultured rat microglia. J Neurochem 1992; 58: 1401-8.
68. Yao J, Keri JE, Taffs RE, Colton CA. Characterization of interleukin-1 by microglia in culture. Brain Res 1992; 591: 88-93.
69. Chao CC, Hu S, Gekker G, Novick WJ, Remington JS, Peterson PK. Effects of cytokines on multiplication of Toxoplasma gondii in microglial cells. J Immunol 1993; 150: 3404-10.
70. Haga S, Ikeda K, Sato M, Ishii T. Synthetic Alzheimer amyloid b/A4 peptides enhance production of complement C3 component by cultured microglial cells. Brain Res 1993; 601: 88-94.
71. Lee SC, Liu W, Dickson DW, Brosnan CF, Berman JW. Cytokine production by human microglia and astrocytes. J Immunol 1993; 150: 2659-67.
72. Bader MF, Taupenot L, Ulrich G, Aunis D, Ciesielski-Treska J. Bacterial endotoxin induces [Ca2+]i transients and changes the organization of actin in microglia. Glia 1994; 11: 336-44.
73. Mizuno T, Sawada M, Marunouchi T, Suzumura A. Produc-tion of interleukin-10 by mouse glial cells in culture. Biochem Biophys Res Commun 1994; 205: 1907-15.
74. Norenberg W, Gebicke-Haerter PJ, Illes P: Voltage-dependent potassium channels in activated rat microglia. J Physiol (Lond) 1994; 475: 15-32.
75. Patrizio M, Levi G. Glutamate production by cultured microglia: differences between rat and mouse, enhance-ment by lipopolysaccharide and lack effect of HIV coat protein gp120 and depolarizing agents. Neurosci Lett 1994; 178: 184-8.
76. Chao CC, Gekker G, Sheng WS, Hu S, Tsang M, Peterson PK. Priming of morphine on the production of tumor necrosis factor-a by microglia: implications in respiratory burst activity and human immunodeficiency virus-1 expression. J Pharmacol Exp Ther 1994; 269: 198-203.
77. Abd-El-Basset E, Fedoroff S. Effect of bacterial wall lipopo-lysaccharide on morphology, motility, and cytoskeletal organization of microglia in cultures. J Neurosci Res 1995; 41: 222-37.
78. Appel K, Buttini M, Sauter A, Gebicke-Haerter PJ. Cloning of rat interleukin-3 receptor b-subunit from cultured microglia and its mRNA expression in vivo. J Neurosci 1995; 15: 5800-5809.
79. Gottschall PE, Yu X, Bing B. Increased production of gelatinase B (matrix metalloproteinase-9) and interleukin-6 by activated rat microglia in culture. J Neurosci Res 1995; 42: 335-42.
80. Hayashi M, Luo Y, Laning J, Strieter RM, Dorf ME. Production and function of monocyte chemoattractant protein-1 and other b-chemokines in murine glial cells. J Neuroimmunol 1995; 60: 143-50.
81. Kim YS, Kennedy S, Tauber MG. Toxicity of Streptococcus pneumoniae in neurons, astrocytes, and microglia in vitro. J Infec Dis 1995; 171: 1363-8.
82. Minghetti L, Levi G. Induction of prostanoid biosynthesis by bacterial lipopolysaccharide and isoproterenol in rat microglial cultures. J Neurochem 1995; 65: 2690-8.
83. Patrizio M, Costa T, Levi G. Interferon-g and lipopoly-saccharide reduce cAMP responses in cultured glial cells: reversal by a type IV phosphodiesterase inhibitor. Glia 1995; 14: 94-100.
84. Ryan RE, Sloane BF, Sameni M, Wood PL. Microglial cathepsin B: an immunological examination of cellular and secreted species. J Neurochem 1995; 65: 1035-45.
85. Sakai N, Kaufman S, Milstien S. Parallel induction of nitric oxide and tetrahydrobiopterin synthesis by cytokines in rat glial cells. J Neurochem 1995; 895-902.
86. Sawada M, Suzumura A, Marunouchi T. Induction of functional interleukin-2 receptor in mouse microglia. J Neurochem 1995; 64: 1973-9.
87. Walker DG, Kim SU, McGeer PL. Complement and cytokine gene expression in cultured microglia derived from postmortem human brains. J Neurosci Res 1995; 40: 478-93.
88. Fincher IV EF, Johannsen L, Kapas L, Takahashi S, Krueger JM. Microglia digest Staphylococcus aureus into low molecular weight biologically active compounds. Am J Physiol 1996; 271: R149-R156.
89. Lodge PA, Sriram S. Regulation of microglia activation by TGF-b, IL-10, and CSF-1. J Leukoc Biol 1996; 60: 502-508.
90. Zhang SC, Fedoroff S. Neuron-microglia interactions in vitro. Acta Neuropathol 1996; 91: 385-95.
91. Iglesias BM, Cerase J, Ceracchini C, Levi G, Aloisi F. Analysis of B7-1 and B7-2 costimulatory ligands in cultured microglia: upregulation by interferon-g and lipopolysaccha-ride and downregulation by IL-10, PGE2 and cAMP-elevating agents. J Neuroimmunol 1997; 72: 83-93.
92. Zuckerman SH, Gustin J, Evan GF. Expression of CD54 (Intercellular adhesion molecule-1) and the b1 integrin CD29 is modulated by a cyclic AMP dependent pathway in activated primary rat microglial cell cultures. Inflammation 1998; 22: 95-106.
93. Mayer AMS, Oh S, Ramsey KH, Romanic A. Escherichia coli lipopolysaccharide appears to induce apoptosis in rat brain microglia. Soc Neurosci Abstr 1998 (in press).
94. Yoshida T, Tanaka M, Sotomatsu A, Hirai S. Activated microglia cause superoxide-mediated release of iron from ferritin. Neurosci Lett 1995; 190: 21-4.
95. Woodroofe MN, Hayes GM, Cuzner ML. Fc receptor density, MHC antigen expression and superoxide produc-tion are increased in interferon-gamma-treated microglia isolated from adult brain. Immunology 1989; 68: 421-6.
96. Burge T, Griot C, Vandevelde M and Peterhans E. Antiviral antibodies stimulate production of reactive oxygen species in cultured canine brain cells infected with canine distemper virus. J Virol 1989; 63: 2790-7.
97. Suzumura A, Marunouchi T, Yamamoto H. Morphological transformation of microglia in vitro. Brain Res 1991; 545: 301-6.
98. Piani D, Spranger M, Frei K, Schaffner A, Fontano A. Macrophage-induced cytotoxicity of N-methyl-D-aspartate receptor postive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur J Immunol 1992; 22: 2429-36.
99. Chao CC, Hu S, Molitor TW, Shaskan EG, Peterson PK. Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J Immunol 1992; 149: 2736-41.
100. Colton CA, Yao J, Keri JE, Gilbert D. Regulation of microglial function by interferons. J Neuroimmunol 1992; 30: 89-98.
101. Zielasek J, Tausch M, Toyka KV, Hartung HP. Production of nitrite by neonatal rat microglial cells/brain macrophages. Cell Immunol 1992; 141: 111-20.
102. Banati RB, Schubert P, Rothe G, et al. Modulation of intracellular formation of reactive oxygen intermediates in peritoneal macrophages and microglia/brain macrophages by propentofylline. J Cereb Blood Flow Metab 1994; 14: 145-9.
103. Corradin SB, Mauel J, Donini SD,Quattrocchi E, Ricciardi-Castagnoli P. Inducible nitric oxide synthase activity of cloned murine microglial cells. Glia 1993; 7: 255-62.
104. Klegeris A, McGeer PL. Rat brain microglia and peritoneal macrophages show similar responses to respiratory burst stimulants. J Neuroimmunol 1994; 53: 83-90.
105. Colton CA, Jia M, Li MX, Gilbert DL. K+ modulation of microglial superoxide production: involvement of voltage-gated Ca2+ channels. Am J Physiol 1994; 266: C1650-C1655.
106. Colton CA, Snell J, Chernyshev O, Gilbert DL. Induction of superoxide anion and nitric oxide production in cultured microglia. Ann N Y Acad Sci 1994; 738: 54-63.
107. Chao CC, Hu S, Peterson PK. Modulation of human microglial superoxide production by cytokines. J Leukoc Biol 1995; 58: 65-70.
108. Hu S, Chao CC, Khanna KV, Gekker G, Peterson PK, Molitor TW. Cytokine and free radical production by porcine microglia. Clin Immunol Immunopathol 1996; 78: 93-6.
109. Ii M, Sunamoto M, Ohnishi K, Ichimori Y. b-amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity. Brain Res 1996; 720: 93-100.
110. Janabi N, Chabrier S, Tardieu M. Endogenous nitric oxide activates prostaglandin F2á production in human microglial cells but not in astrocytes. J Immunol 1996; 157: 2129-35.
111. Mohan PF, Ard MD. Induction of microglial nitric oxide synthesis by very low density lipoprotein. Glia 1996; 17: 259-62.
112. Mosley K, Cuzner ML. Receptor-mediated phagocytosis of myelin by macrophages and microglia: effect of opsoni-zation and receptor blocking agents. Neurochem Res 1996; 21: 481-7.
113. Van Muiswinkel FL, Veerhuis R, Eikelenboom P. Amyloid b protein primes cultured rat microglial cells for enhanced phorbol 12-myristate 13-acetate-induced respiratory burst activity. J Neurochem 1996; 66: 2468-76.
114. Colton C, Wilt S, Gilbert D, Chernyshev O, Snell J, Dubois-Dalq M. Species differences in the generation of reactive oxygen species by microglia. Mol Chem Neuropathol 1996; 28: 15-20.
115. Si QS, Nakamura Y, Kataoka K. Albumin enhances superoxide production in cultured microglia. Glia 1997; 21: 413-8.
116. Si QS, Nakamura Y, Kataoka K. Hypothermic suppression of microglial activation in culture: inhibition of cell proliferation and production of nitric oxide and superoxide. Neuroscience 1997; 81: 223-9.
117. Murata JI, Castagnoli P, Mange P, Martin F, Jeanneret LJ: Microglial cells induce cytotoxic effects toward colon carcinoma cells: measurement of tumor cytotoxicity with a ã-glutamyl transpeptidase assay. Int J Cancer 1997; 70: 169-74.
118. Klegeris A, McGeer PL. b-amyloid protein enhances macrophage production of oxygen free radicals and glutamate. J Neurosci Res 1997; 49: 229-35.
119. Ding M, St. Pierre BA, Parkinson JF, et al. Inducible nitric-oxide synthase and nitric oxide production in human fetal astrocytes and microglia. J Biol Chem 1997; 272: 11327-35.
120. Bhat NR, Zhang P, Lee JC, Hogan EL. Extracellular signal-regulated kinase and p38 subgroups of MAPK regulate inducible nitric oxide synthase and TNF-a gene expression in endotoxin-stimulated primary glial cultures. J Neurosci 1998; 18: 1633-41.
121. Lockhart BP, Cressey KC, Lepagnol JM. Suppression of nitric oxide formation by tyrosine kinase inhibitors in murine N9 microglia. Br J Pharmacol 1998; 123: 879-89.
122. Orskov I, Orskov F, Jann B, Jann K. Serology, chemistry and genetics of O and K antigens of Escherichia coli. Bacteriol Rev 1977; 41: 667-710.
123. Haber F, Weiss J. The catalytic decomposition of hydrogen peroxide by iron salts. Proc R Soc Lond A 1934; 147: 333-51.
124. Imlay JA, Linn S. DNA damage and oxygen radical toxicity. Science 1988; 240: 1302-9.
125. Beckman JS. Peroxynitrite versus hydroxyl radical: The role of nitric oxide in superoxide-dependent cerebral injury. Ann N Y Acad Sci. 1994; 738: 69-75.
126. Babior B. Oxidants from phagocytes: agents of defense and destruction. Blood 1984; 64: 959-66.
127. Neugebauer E, Lechleuthner A, Rixen D, Saad S. Pharmacotherapy of shock. In: Chernow B (ed). The Pharmacologic approach to the critically ill patient. Williams & Wilkins, 1994; 1104-21.
128. Weikert LF, Bernard GR. Pharmacotherapy of Sepsis. Clin Chest Med 1996; 289-305.
129. Galley HF, Howdle PD, Walker BE, Webster NR. The effects of intravenous antioxidants in patients with septic shock. Free Radic Biol Med 1997; 23: 768-74.
130. Pfister HW, Scheld WM. Brain injury in bacterial meningitis: therapeutic implications. Curr Opin Neurol 1997; 10: 254-9.
131. Frohlich L, Riederer P. Free radical mechanisms in dementia of Alzheimer type and potential for antioxidative treatment. Arzneimittelforschung 1995; 45: 443-6.
132. Mayer AMS, Spitzer JA: Modulation of superoxide anion generation by manoalide, arachidonic acid and staurosporine in liver infiltrated neutrophils in a rat model of endotoxemia. J Pharmacol Exp Ther 1993; 267: 400-9.
133. Mayer AMS, Brenic S, Stocker R, Glaser KB. Modulation of phorbol 12-myristate 13-acetate stimulated O2- generation in in vivo lipopolysaccharide-primed rat alveolar macropha-ges by protein kinase C, phospholipase A2, protein serine-threonine phosphatases(s), protein tyrosine kinase(s) and phosphatase(s) inhibitors and arachidonic acid. J Pharmacol Exp Ther 1995; 274: 427-36.
134. Mayer AMS, Brenic S, Glaser KB. Pharmacological targeting of signalling pathways in protein kinase C-stimulation superoxide generation in neutrophil-like HL-60 cells: Effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s), phospholipase A2. J Pharmacol Exp Ther 1996; 279: 633 -44.
135. Heppner FL, Roth K, Nitsch R, Hailer NP. Vitamin E induces ramification and downregulation of adhesion molecules in cultured microglial cells. Glia 1998; 22: 180-8.
136. Mayer AMS, Oh S. Pharmacological targeting of signaling pathways in phorbol ester-stimulated superoxide genera-tion in LPS-primed rat brain microglia. Soc Neurosci Abstr 1997; 23(2): 1443.
137. Oh S, Mayer AMS. The marine natural product manoalide inhibits superoxide and thromboxane B2 production by Escherichia coli LPS-activated rat brain microglia. The Faseb J. 1998:12(4): A461.

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