T H E J O U R N A L OF

PEDIATRICS MAY

1990

Volume 116

Number 5

MEDICAL PROGRESS We believethat this reviewof the molecular pathophysiologyof meningitiswill serve also as a reviewof what is known about the inflammatory process in general. We suspect that terms such as interleukin, cytokine, and tumor necrosisfactor will be seen more commonly in the pediatric literature. The hope is that better understanding of the underlying processes will lead to more specific and effective therapy.--J.M.G.

Molecular pathophysiology of bacterial meningitis: Current concepts and therapeutic implications Xavier SOez-Llorens, MD, O c t a v i o Ramilo, MD, M a h m o u d M. Mustafa, MD, Jussi Mertsola, MD, a n d G e o r g e H. M c C r a c k e n , Jr., MD From the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas

Despite advanced medical intensive care technology and the availability of active antimicrobial agents for the treatment of bacterial meningitis, the case fatality and long-term morbidity rates have not appreciably changed. Current case fatality rates are 3% to 7% in infants and children, increasing to 30% in neonates and adults. Long-term neurologic sequelae develop in as many as one third of all survivors. Several groups of investigators have attempted to elucidate the molecular pathophysiology of bacterial meningitis, a complex process that encompasses the bacterial components that initiate events, the participation of mediators that elicit the meningeal inflammatory response, and the altered physiology in the brain, including increased pressure and reduced blood flow, that can result in neurologic sequelae.

Reprint requests: Xavier Sfiez-Llorens,MD, Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9063. 9/18/19575

In this review we will describe each of the currently identified protagonists of the complex meningeal inflammatory network, the experimental meningitis models and the clinical studies in which they have been identified, the possible targets for therapeutic intervention, and the results of recently conducted clinical trials of dexamethasone therapy for bacterial meningitis. P R O T A G O N I S T S OF T H E M E N I N G E A L INFLAMMATORY CASCADE Animal models of meningitis have provided substantial information on the pathogenesis and pathophysiology of this disease. Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, Escherichia coli K1, and several other microorganisms have been used to induce meningitis in rabbits or rats. 18 Active components of these bacteria have been identified in these animal models. The roles of leukocytes and other host defense mechanisms, and of several inflammatory mediators, have been subjects of extensive research. Although these models have provided

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BBBP Blood-brain barrier permeability CSF Cerebrospinal fluid DXM Dexamethasone Hib Haemophilus influenzae type b 1L-13 Interleukin- 1 beta LOS Lipooligosaccharide LPS Lipopolysaccharide PAF Platelet-activating factor PGE2 Prostaglandin E2 TNF Tumor necrosis factor TNFc~ Tumor necrosis factor alpha insights concerning these potential protagonists, the relevance of these events to those which occur in human subjects with bacterial meningitis has only recently been investigated. Bacterial components. Three components of bacteria have been implicated in the virulence of the most common organisms causing meningitis: the capsule, the cell wall, and lipopolysaccharide. The capsule. H. influenzae type b, S. pneumoniae, E. coli K1, and N. meningitidis synthesize large amounts of a condensed, well-defined extracellular layer of polysaccharide that surrounds the cell. This capsule contributes to the invasiveness of these microorganisms by facilitating their evasion of host recognition and clearance. 9-11 There is no evidence, however, that the capsule of these meningeal pathogens has a direct role in inducing changes in bloodbrain barrier permeability or inflammation within the subarachnoidal space. 12, 13 The cell wall. The principal difference between grampositive and gram-negative bacteria resides in the components of their cell walls. The cell wall of gram-positive organisms contains many peptidoglycan layers, composing up to 90% of the cell wall, whereas in gram-negative bacteria there is usually only one layer that constitutes 5% to 20%. The glycan-peptide network of S. pneumoniae induces meningeal inflammation when inoculated intracisternally into rabbits. 14' 15 Most cell walls of gram-positive bacteria contain considerable amounts of teichoic acid, which can constitute up to 50% of the dry weight of the wall; this substance is absent in gram-negative bacteria. This water-soluble polymer can be found in two forms: the wall teichoic acid covalently linked to peptidoglycan, and the membrane teichoic acid (lipoteichoic acid) covalently bound to membrane glycolipids (membrane-associated Forssman antigen). The teichoic acid polymers of S. pneumoniae are strong inducers of meningeal inflammation, t4, 15 Treatment of S. pneumoniae with ampicillin results in cell wall degradation and release of these cell wall components, which, in turn, stimulate a brisk but transient enhancement of inflammation. 16 Confirmation of the inflammatory properties of these components comes from studies of defective

The Journal of Pediatrics May 1990

pneumococci that lack an autolytic system and thus do not lyse after penicillin treatment 17, 18; meningitis caused by these organisms is relapsing in course but is associated with scant inflammatory changes in the cerebrospinal fluid. 18 The lipopolysaccharide. Gram-negative bacteria have attached to their outer membrane LPS molecules consisting of a complex toxic lipid called lipid A, to which is linked a polysaccharide that comprises a core and a terminal series of repeat units. This compound is firmly bound to the cell surface and is released when the cells are lysed. Endotoxin of Hib organisms has been used in experimental meningitis studies to elicit CSF inflammatory changes. The LPS of Hib has a shorter saccharide chain than does classic LPS of enteric bacteria and therefore has been referred to as lipooligosaccharide.19 Once Hib organisms are present in CSF, LOS is released from dying Hib cells as well as from growing cells, mainly in the form of blebs or outer membrane vesicles.2~The intracisternal inoculation of Hib LOS in its purified form or as OMV into rabbits or rats has resulted in dose- and time-dependent increases in BBBP and in the CSF inflammatory response.13, 21-23Preincubation of LOS with polymyxin B, a cationic antibiotic that neutralizes LOS by binding to its lipid A region, 24 or with acyloxyacyl hydrolase, a neutrophil enzyme that deacylates LOS by cleaving fatty acyl chains from lipid A, 25 reduces meningeal inflammation and increased BBBP.! 3,21"23 Considerable evidence indicates that initiation of antibiotic therapy results in increased concentrations of bacteria-free LPS in the CSF of animals with experimental meningitis, 26, 27 most likely as a result of bacterial lysis (Fig. 1). This increment in endotoxin concentration in CSF has been correlated with a rapid increase in the meningeal inflammatory response. Chloramphenicol, a protein synthesis inhibitor, does not cause a significant increase of free endotoxin concentration in the CSF during experimental E. coli meningitis, 26 but we were able to document, shortly after treatment of experimental Hib meningitis, a substantial increase in endotoxin concentration followed by an augmented CSF inflammatory response. 28 Recently, two reports 29,3~ have demonstrated increased endotoxin concentrations in CSF and ventricular fluid of infants with either Hib or coliform meningitis treated with ceftriaxone or with intraventricular gentamicin, respectively. This increment was associated with augmented meningeal inflammation and with an adverse outcome of disease in infants with coliform meningitis who had received gentamicin intraventricularly. 3~ Inflammatory mediators. Although bacterial components could cause some of the central nervous system toxic effects, recent evidence implicates a large, and still growing, family of cytokines that act as critical inflammatory mediators in the local response to bacterial meningitis. Prominent among these cytokines are tumor necrosis factor alpha (cachectin)

Volume l 16 Number 5

and interleukin-1, two monocyte-macrophage hormones secreted in response to microbial or immunologic insuits.31.32 Both proteins stimulate vascular endothelial cells to induce adhesion and passage of neutrophils into the CNS and other tissues, and trigger inflammatory processes. 33, 34 Additionally, several other substances, such as plateletactivating factor, 35 arachidonic acid metabolites, 36 and other interleukins, 37 participate in the complex inflammatory processes that occur in the subarachnoid space. Tumor necrosis factor. The cytokine TNFc~ is a protein that is primarily produced by macrophages and monocytes in response to multiple stimuli. 31 A lymphocyte-derived TNF~ (originally termed lymphotoxin) has many properties in common with TNFc~. Both proteins share a number of functional activities, bind to a similar receptor, are approximately 30% homologous at the protein level, and play an important role in various aspects of immune responsiveness. 3s Because TNFfl has not been extensively studied, it will not be a subject of our discussion. Human TNFc~ is encoded by a gone located on the short arm of chromosome 6. Although monocyte-macrophage cells seem to be the most important sources of TNFc~, other activated cells, including lymphocytes, natural killer cells, Kupffer cells, synovial phagocytes, and, more importantly for the focus of this review, astrocytes and microglial cells of the brain have been shown to produce TNFc~. 39 Depending on concentration, duration of cell exposure, and interaction with other mediators in the cellular environment, the net biologic effects of this regulatory factor may ultimately benefit or injure the host. 39 In physiologic concentrations, TNFc~ is believed to arm the immune system and to assist in tissue healing. In pathologic concentrations, it plays a seminal role in fatal endotoxemia. Administration of TNFc~ to laboratory animals causes physiologic and pathologic changes similar to those observed in animals with gram-negative septicemia.4~ Experimental studies 4143 in animals and human volunteers given endotoxin intravenously demonstrate a rapid rise in serum TNFc~ activity that peaks in approximately 2 hours and is undetectable after 4 to 5 hours. Simultaneous administration of anti-TNFa monoclonal antibodies reduces serum activity and ablates symptoms referable to the endotoxin. 44 TNFc~ can also be detected in sera of patients with meningococcemia, and its concentration appears to be related to severity of illness.45 In experimental meningitis, TNFc~ activity is first detected in the CSF of rabbits 45 minutes after intracisternal inoculation of Hib LOS, peaks at 2 hours, and persists for approximately 5 hours, a response almost identical to that seen in human subjects given endotoxin intrave.nously.46 The presence of CSF pleocytosis is observed 75 minutes after T N F is detected and peaks 6 to 9 hours after endotoxin challenge. Changes in protein, glucose, arid lactate concen-

Molecular pathophysiology of bacterial meningitis

:~

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Fig. I. Mean concentrations of Hib organisms (open bars) and LOS (black bars) in CSF of untreated rabbits (upper panel) and in animals that received ceftriaxone for at 6 hours (lower panel). (Data from Mertsola J, Ramilo O, Mustafa MM, Saez-Llorens X, Risser RC, McCracken Gh Jr. Pediatr Infect Dis J 1989;8:904-6.)

trations in CSF are also observed 3 hours after LOS administration. Sera obtained simultaneously with CSF samples have no detectable TNFc~ activity. Moreover, simultaneous intracisternal administration of anti-TNFa polyclonal antibody with Hib LOS neutralizes CSF T N F a activity and is associated with substantial attenuation of the meningeal inflammatory changes. 46 When live Hib (1 or 2 X 107) organisms are used to induce meningitis, a similar peak T N F a concentration is observed at 3 hours after intracisternal challenge, but activity persists for approximately 14 hours. The prolonged presence of T N F a activity in the C S F of these rabbits is probably best explained by the continuous stimulation of incoming monocytes, macrophages, or equivalent brain cells, because of persistent endotoxin in CSF from multiplying and dying bacteria. Ceftriaxone or chloramphenicol treatment of Hib-induced meningitis in rabbits is associated with a brisk release of bacteria-free endotoxin into CSF from rapidly lysed organisms, and with detection of significantly larger concentra-

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tions of T N F a than those found in untreated animals. 27, 28 The presence of T N F a has been detected in the initial CSF sample of 25 (93%) of 27 neonates with gram-negative enteric bacillary meningitis,47 and in 79 (75%) of 106 infants and children with meningitis caused by Hib, S. pneumoniae, N. meningitidis, or Streptococcus agalactiae. 48 In contrast, T N F a is not found in infants with culture-proved viral meningitis or in CSF from infants without known infection who required CSF examinations for other reasons. 49 Similar results were reported by Nadal et al. 5~ Interleukin-1. The cytokine IL-1 is thought to be one of the principal mediators of the body's response to microbial invasion32 and to be responsible for the induction of fever, changes in leukocyte counts, and synthesis of acute-phase reactants that follow acute bacterial infections. 51 Formerly called "endogenous pyrogen," IL-1 is produced mainly by, and is released from, circulating activated mononuclear phagocytes in response to inducing agents, including microorganisms and their products such as endotoxins, exotoxins, peptidoglycans, and teichoic acids. 32, 52, 53 Two bioehemically distinct but structurally related IL-1 molecules have been cloned in human beings, as well as in other species: I L - l a and IL- 1/3.32 Each human IL-1 is coded by a separate gene, but both are located on chromosome 2. The amino acids of the interleukins are only 26% homologous. IL- 1/3 is more prevalent in body fluids, but both types are thought to have the same biologic effects. Besides monocyte-macrophage cells, the synovial fibroblasts, epidermal cells, vascular endothelial cells, neutrophils, and astrocytes and microglial cells of the brain 32 also produce IL-1. Activity of IL-1/3 can be detected in the CSF of rats 30 minutes after intracisternal injection of Hib LOS, and its concentration is significantly decreased by 2 hours after inoculation. 54 Data from our laboratory indicate that recombinant rabbit tL-1/3, inoculated directly into the CSF of rabbits, is capable of inducing significant alteration of meningeal inflammation in a dose-dependent fashion. 55 The inflammatory changes induced by recombinant rabbit IL- 1/3 are delayed in comparison with those induced by purified Hib LOS. Additionally, T N F a activity is not detected in CSF of rabbits with recombinant rabbit IL-113-induced meningitis. Inoculation of specific polyclonal antibodies against recombinant rabbit IL-li3 simultaneously with recombinant rabbit IL-I/3 results in almost complete suppression of the inflammatory response. Similarly, other investigators induced dose-dependent meningeal inflammation and BBBP changes when human recombinant IL-1/3 was inoculated intracisternally into adult rats. 56 Again, preincubation of human recombinant IL-1/3 with a monoclonal antibody against it significantly inhibits those alterations. Although intracisternal administration of human

The Journal of Pediatrics May 1990

T N F a does not induce meningitis in rats, its combination with human recombinant IL-1/3 was thought to be synergistic in provoking meningeal inflammation and alterations of BBBP. 56 Natural rabbit T N F a does induce a prompt meningeal inflammatory response when administered intracisternally to rabbits; this reaction is prevented by mixing the T N F a with its monoclonal antibody before injection (authors' unpublished data). Activity of IL-1/3 can be detected in initial CSF samples of almost all neonates, 47 infants, and children 48 with bacterial meningitis caused by the common meningeal pathogens, and its presence is significantly correlated with CSF inflammatory abnormalities, T N F concentrations, and adverse outcome. Neurologic sequelae are associated with IL-1/3 concentrations >500 pg/ml in CSF obtained at the time of diagnosis. Infants with culture-proved viral meningitis or with normal CSF have low or nondetectable IL-1/3 concentrations. These data indicate a prominent role of IL-113 in the initial events of meningeal inflammation, but its specific place in the inflammatory cascade and its relation to other cytokines, such as TNF, remain to be defined. Phospholipase A2-induced substances. Two important inflammatory pathways are generated by the enzymatic activity of phospholipase A2 on membrane phospholipids: the glycerophosphocholine pathway, in which PAF is the prin. cipal product, 57 and the arachidonic acid pathway, in which the cyclooxygenase products (prostaglandins and thromboxanes) and lipooxygenase products (leukotrienes) are produced. 58 All these lipid autacoids have the collective ability to orchestrate many aspects of the inflammatory process by way of primary, secondary, or synergistic actions. They are rapidly released by a variety of cells, including neutrophils, platelets, and vascular endothelial cells, after stimulation with bacterial and immunologic antigens. Evidence suggests that cytokines such as T N F and IL- 1 induce phospholipase A2 activity and therefore trigger production of these lipid proinflammatory substances. 57, 58 PLATELET-ACTIVATINGFACTOR.P A F is an endogenously formed, potent glycerophosphocholine derivative with a myriad of biologic activities.59 Many of the physiologic and pathologic effects induced by P A F are similar to those observed after endotoxin exposure. 57 After intravenous administration to animals, P A F causes aggregation of platelets, neutrophils, and monocytes, provoking diminished concentrations of these cells in blood and thrombotic phenomena within vascular compartments. In vitro, P A F induces chemotaxis, degranulation, and adherence of polymorphonuclear cells to the endothelium; increases vascular permeability; and triggers the coagulation cascade. In addition, when PAF is inoculated intracutaneously, in picomolar concentrations, into human volunteers, a severe necrotizing vasculitis is observed. 6~

Volume 116 Number 5 There is scant information regarding the specific role of pAF in local or disseminated bacterial infections. Arditi et al.35 found significantly elevated PAF concentrations in CSF of 15 infants with Hib meningitis compared with control patients. The specific role of PAF in the pathogenesis of bacterial meningitis and its relation to other inflammatory mediators is uncertain. ARACHIDONICACIDMETABOLITES.Of the various arachidonic acid metabolites formed by phospholipase A2 activity on membrane phospholipids, the prostaglandins E2 and I2 (prostacyclin) and leukotriene B4 are potent mediators of inflammation,s8 Cellular events mediated by leukotriene B4 include activation of human neutrophils for self-adherence (aggregation), induction of chemotaxis, and stimulation of enzyme release on degranulation. Although leukotrienes have been linked to potent proinflammatory activities in various tissues, their role in the pathogenesis of bacterial meningitis, if any, is not established. Increased amounts of PGE2 in the CSF of rabbits can be demonstrated several hours after intracisternal challenge with live pneumococcus or its purified cell wall, and a positive correlation can be observed between the concentration of PGE2 and the number of leukocytes in CSF and changes in blood flow and vascular permeability.16' 61 Additionally, antibiotic treatment of rabbits with pneumococcal meningitis causes significantly higher CSF PGE2 concentrations than those found in saline solution-treated animals. Reduction of CSF PGE2 concentrations with indomethacin, however, is associated with increased CSF leukocytosis compared with that in untreated rabbits; however, cerebral edema is attenuated. 61 Recently, Kadurugamura et al. 62 demonstrated no CSF pleocytosis for up to 6 hours after PGE2 was intracisternally administered to rabbits. There was, however, a dose-related increase in protein content at 2 hours after prostaglandin administration. These data suggest that PGEz is not a leukocyteehemoattractant and that it may down-regulate other inflammatory mediators that induce CSF pleocytosis. Recent in vitro studies indicate that PGE2 and prostacyclin control production of monocyte-produced TNFc~ at the level of messenger RNA, thereby acting in a classic feedback inhibitory manner. 63 Selective blockage of PGE2 with cyclooxygenase inhibitors may possibly be associated with increased CSF T N F concentration and worsening of some inflammatory indexes. Detectable concentrations of PGE2 and prostacyclin can be measured in the initial CSF sample of approximately 90% and 50%, respectively, of infants and children with bacterial meningitis. 36 High concentrations of PGE2 correlate directly with IL-1, prostacyclin, leukocyte, lbrotein, and lactate concentrations in CSF and with duration of fever and incidence of neurologic sequelae;, there is an inverse correlation with CSF glucose concentrations.

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On the basis of these experimental and clinical data, it appears that prostaglandins participate in the inflammatory events that occur in bacterial meningitis. Their specific effects and interactions with other inflammatory cytokines are largely unknown. Other inflammatory mediators. There is accumulating evidence that other substances may also play an important role in the generation of meningeal inflammation. These prointtammatory products include complement factors, 64~65 other interleukins 37,66 (1L-6, IL-8), and maerophage-inflammatory proteins. 66 In addition, other noninflammatory substances (excitatory amino acids) have been implicated in neuronal damage during experimental bacterial meningitis.67, 68 The presence of leukocyte chemotactic activity in C S F has been reported by several investigators; the major component involved is complement factor 5a (C5a).64, 65 In experimental pneumococcal meningitis, C5a-derived chemotactic activity appears to be responsible for the early influx of leukocytes into CSF. When rabbits are depleted of complement by intravenous administration of cobra venom, there is a delay in the appearance of leukocytes in CSF. 69 By contrast, arachidonic acid metabolites and other substances seem to be responsible for late recruitment of neutrophils in the absence of complement. 7~This concept is not clear, because cobra venom factor depletes C3 by way of its C3-cleaving capacity but is inactive against C5. 71 When C5a is administered intracisternally to rabbits, there is a rapid influx of leukocytes into CSF that peaks 1 hour after challenge and persists for 6 hours. 62 Coadministration of PGE2 with C5a decreases CSF leukocytosis in a doserelated fashion. Additional studies are required to define the specific role of complement factors in the pathogenesis of bacterial meningitis. Formerly called hepatocyte-stimulating factor or interferon beta-2, IL-6 is a peptide considered the most potent inducer of acute-phase protein reactants in response to bacterial infections. 72 This acute reaction is characterized by fever, leukocytosis, increase in erythrocyte sedimentation rate, increase in secretion of corticotropin and glucocorticoids, activation of complement and the clotting cascade, and increase in concentration of C-reactive protein. IL-6 is produced by a variety of cells, including monocytes, endothelial cells, and astrocytes, primarily in response to IL-1 stimulation. Although IL-6 can be detected in the CSF of infants and children with bacterial meningitis, its presence is not correlated with any of the indexes of meningeal inflammation or with severity of disease. 37 Recently discovered, the family of macrophage-inflammatory proteins (types 1 and 2) are being intensively studied for their proinflammatory properties and strong neutrophit chemoattractant actions. 73'74 They are also produced by

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different cells after endotoxin or cytokine stimulation. Macrophage-inflammatory protein type 2 has structural and biologic similarities with a neutrophil-activating peptide that is currently called interleukin-8. 75 It appears that the macrophage-inflammatory proteins types 1 and 2 are also capable of inducing CSF pleocytosis, increased BBBP, and brain edema when injected intracisternally into rabbits. 66 An important focus of recent research involves investigations of the role of calcium and excitatory amino acids (glutamate and aspartate) in the pathogenesis of brain injury.67, 68 These amino acids are associated with cell excitation through the opening of the receptor-operated calcium channels. The subsequent intracellular accumulation of calcium produces profound metabolic perturbations leading to cell swelling (brain edema) and neuronal cell death. These neurotoxic excitatory amino acids, especially glutamate, may have a potential role in the pathogenesis of brain damage from meningitis, because preliminary evidenc e suggests that they increase substantially in the CSF of rabbits with experimental meningitis induced by endotoxin, pneumococcal cell walls, or live Pseudomonas aeruginosa. 68 Moreover, glutamate concentrations are closely correlated with lactate concentrations in CSF. In summary, the emerging understanding of the role of inflammatory mediators in the meningeal inflammatory resPonse, and of their interaction with other substances to amplify the host response after microbial invasion, provides new strategies for potentially effective therapeutic interventions to regulate inflammation and possibly to prevent brain damage. Polymorphonuclear neutrophil leukocytes. Neutrophils are the predominant phagocytes of circulating blood and play a central role in host defense. 76 Despite being an effective defense against bacterial pathogens, neutrophils also act as mediators of tissue-destructive events in many inflammatory diseases. These harmful activities have been linked to release of a complex assortment of agents that can destroy normal cells and dissolve connective tissues. These agents include a family of reactive oxidizing chemicals and several toxic substances that reside within specific granules. Although these toxins normally defend the host against invading microbes, the neutrophit has little intrinsic ability to distinguish foreign from host antigens and relies on other arms of the immune system (e.g., antibodies, complement, cytokines) to select its targets. 76 Studies in animal model s suggest that polymorphonuclear leukocytes do little to control the infection within the subarachnoid space and, instead, may have detrimental effects on the outcome of bacterial meningitisY, 77-80 Many factors contribute to the poor phagocytosis exhibited by polymorphonuclear leukocytes in the meningeal space, in-

The Journal of Pediatrics May 1990

cluding weak functional activity in a fluid medium, lack of CSF opsonic properties and of complement activity, 81 and poor penetration of antibodies through the blood-brain barrier. 82 Studies suggest that bacterial eradication from CSF is not a leukocyte-dependent phenomenon, because in leukopenic animals the bacterial growth rate and the maximal bacterial concentrations attained in CSF after intracisternal inoculation do not differ from those observed in normal rabbits. 77, 79 In addition, the influx of leukocytes into the CNS can result in deleterious alterations of CSF dynamics, brain metabolism, and CNS homeostasis. 79 One of the initial pathologic events that occurs in bacterial meningitis is thought to be the disruption of the bloodbrain barrier. 12 This alteration in permeability is probably a result of a complex interaction between bacterial components and inflammatory mediators that promote the adherence and transendothelial passage of neutrophils into the CNS. 56The increased neutrophil adherence to endothelium is recognized as a hallmark of the acute inflammatory response, constituting a prerequisite for extravascular migration of neutrophils at sites of inflammation, and also as a possibly critical step in the pathogenesis of vascular injury associated with acute inflammation. 83 In vivo studies provide valuable insights into this neutrophil-endothelium interaction. The endothelium and neutrophils become more adhesive when stimulated by cytokines such as TNF, IL-I, and PAF. 84, 85 In this regard, complement, especially C5a, seems to be important in the early recruitment of leukocytes, 69 whereas other inflammatory substances, including arachidonic acid metabolites and IL-8, are responsible for their late accumulation at sites of infection. 7~ 75 Membrane glycoproteins are present on the endothelium (endothelialleukocyte adhesion molecule 86) and on leukocytes (CD18 family of adhesion-promoting receptors) 87 that are stimulated by different inflammatory cytokines. Once neutrophils adhere to cerebral capillary endothelial cells, emigration through intercellular junctions by enzymatic digestion proceeds, and this process contributes to increased BBBP and influx of serum proteins into CSF. These concepts are supported by results in experimental meningitis models; inhibition of adherence and migration of leukocytes by various therapeutic interventions (e.g., monoclonal antibodies to the adhesion-promoting receptors) is associated with reduced CSF chemical alterations, diminished BBBP, decreased brain edema, increased survival time, and reduced tissue damage. 69 Moreover, rendering animals leukopenic with cyclophosphamide also is correlated with a reduction of CSF abnormalities and brain edema and with improved outcome of disease. 79, 88 Thus it is likely that the polymorphonuclear leukocyte represents one of the principal cellular components in the pathogenesis of meningeal inflammation; its interaction

Volume l 16 Number 5

Molecular pathophysiology o f bacterial meningitis

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with other inflammatory mediators could explain the molecular basis for brain damage that occurs during the course of bacterial meningitis. CURRENT

CONCEPT

PATHOPHYSIOLOGY MENINGITIS

OF THE OF BACTERIAL

In bacterial meningitis (Fig. 2), the mucosal surfaces in the nasopharynx are the initial site of colonization for three common meningeal pathogens: Hib, S. pneumoniae, and N. meningitidis. This event results most commonly in an asymptomatic carrier state, or infrequently the organisms gain access to the bloodstream. Once there, the organisms avoid phagocytosis because of the presence of a polysaccharide capsule and the absence of protective concentrations of antibodies directed against the capsule. 8991 Depending on the number of circulating bacteria, they enter the CNS through vulnerable sites of the blood-brain barrier (choroid plexus or cerebral microvascular). 92 Because of insufficient humoral factors and phagocytic activity in CSF, organisms multiply rapidly and liberate active cell wall or membrane-associated components (endotoxin, teichoic acid). Initial antibiotic treatment results in rapid lysis of bacteria with release of large concentrations of'these active bacterial products into the CSF. These potent inflammatory products stimulate endothelial cells or macrophage-equivalent brain Cells (astrocytes, microglia), or both, to produce

TNFc~, IL-1, and other mediators. The presence of IL-1 can provoke further production of IL-I by the endothelium. 32 These cytokines activate adhesion-promoting receptors on cerebral vascular endothelial cells, resulting in attraction and attachment of leukocytes to sites of stimuli. Endotoxin can also act directly on endothelium. 93, 94 Once attached, leukocytes traverse the intercellular junctions of the cells as a result of enzymatic action of released proteolytic products. Concomitantly, the cytokines activate phospholipase A2, with subsequent formation of P A F and arachidonic acid metabolites from membrane phospholipids of endothelial and polymorphonuclear cells. These inflammatory changes result in injury to the vascular endothelium, resulting in increased BBBP and activation of the coagulation cascade. Depending on the potency and duration of the inflammatory stimuli, the BBBP is altered to different degrees, and serum proteins and other macromolecules penetrate into CSF. This increase in BBBP results in vasogenic edema. 95' 96 Guided by gradient chemotactic stimuli, larger numbers of leuk0cytes enter the subarachnoid spaces and release toxic substances, resulting in cytotoxic edema. 97 Individually or collectively, all these inflammatory events, if not modulated promptly and effectively, will eventually cause alteration of CSF dynamics, brain metabolism, and cerebrosvascular autoregulation. The consequences of these pathologic changes include severe brain edema, increased intracranial pressure, and

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reduced cerebral blood flow. Brain edema may be the result of many different factors acting in concert: inflammatory mediators, granulocytes and their products, excitatory amino acids, obstruction of CSF resorption, and inappropriate secretion of antidiuretic hormone. 9s Increased intracranial pressure results from the sum of the total brain volume, intracranial blood flow, and CSF volume; therefore all types of edema (vasogenic, cytotoxic, interstitial), plus increased CSF viscosity caused by the blocking by polymorphonuclear leukocyte exudate of free CSF circulation and resorption, are responsible for augmented intracranial pressure. 99-1~ Cerebral blood flow is closely related to intracranial pressure changes during the acute phase of infection. Significant increases in intracranial pressure result in a decrease in cerebral perfusion pressure. 1~ 102 The resultant hypoxemia causes anaerobic metabolism, increasing lactate concentrations and consumption of glucose (hypoglycorrhachia). In severely affected patients, vascular autoregulation in the CNS can be ablated, causing cerebral blood flow to become dependent solely on systemic arterial pressure. 103Cerebral blood flow is normally constant within a range of systemic blood pressures from 60 to 170 mm Hg. When autoregulation is disturbed, systemic hypotension results in decreased cerebral blood flow and brain tissue ischemia. In addition, vasculitis and thrombotic phenomena may result in ischemic infarction of the brain, reducing further the cerebral blood flow. The interaction of all of these events will eventually lead to neuronal injury and to the irreversible focal or diffuse brain damage that occurs commonly after bacterial meningitis. T H E R A P E U T I C A P P R O A C H E S TO REDUCE MENINGEAL INFLAMMATION Understanding the molecular basis of the pathophysiologic changes in bacterial meningitis is crucial to identify potential targets for adjunctive therapy. Potential therapeutic interventions can be directed against harmful bacterial products (cell wall fragments, endotoxin), inflammatory mediators (TNFa, IL-1, PAF, arachidonic acid metabolites), leukocyte activation (adherence, migration, degranulation), or pathophysiologic consequences of the disease (brain edema, intracranial pressure, cerebral blood flow). In this section, we will discuss four therapeutic agents that have been studied in experimental models and their potential applicability in clinical settings. Nonsteroldai antiinflammatory drugs. The antiinflammatory properties of nonsteroidal antiinflammatory drugs reside exclusively in their ability to inhibit the conversion of arachidonic acid to biologically active prostaglandins. 39 Indomethacin, the prototype agent, has been the most thoroughly studied of these drugs in experimental meningitis models. It appears to inhibit cyclooxygenase activity by

The Journal of Pediatrics May 1990

binding in a stereospecific manner to one or another subunit of the enzyme. Because of their selective enzymatic activity, the nonsteroidal antiinflammatory drugs are not expected to interfere with the production of PAF or with the formation of leukotrienes. In experimental pneumococcal meningitis in rabbits, indomethacin treatment blocks development of brain edema in both gray and white matter, but unlike dexamethasone, it does not affect intracranial hypertension. 62 In those experiments, CSF PGE2 concentrations were significantly reduced in animals treated with indomethacin. In the same model, oxindanac, a potent investigational cyclooxygenase inhibitor, reduced the leakage of serum proteins into the CSF and CSF PGE2 concentrations, reduced mortality rates, and decreased clinically evident neurologic sequelae in a large number of rabbits compared with untreated animals. 1~ Indomethacin treatment, however, was associated with an increased number of leukoeytes in CSF. This result can be explained, at least in part, by the fact that PGE2 is an antichemoattractant for leukocytes, probably because of its regulatory effect on TNF production. Additional studies in animals are required before nonsteroidal antiinflammatory drugs can be tested in clinical trials of bacterial meningitis. Pentoxifylline. Pentoxifylline is a phosphodiesterase inhibitor (methylxanthine derivative) that affects functional properties of polymorphonuclear leukocytes once they are activated by endotoxin or cytokines.105 Polymorphonuclear leukocytes, activated by TNF or IL-1 and preincubated with pentoxifylline, show decreased adherence to endothelial cells, decreased production of superoxide and other oxygen radicals, and a reduced rate of granule enzyme release and of liberation of proteolytic products. The exact mechanism of the antiinflammatory activities of pentoxifylline is unclear, but this drug increases intracellular cyclic adenosine monophosphate concentrations, thereby decreasing intracellular accumulation of calcium and activation of polymorphonuclear leukocytes. As a probable result of increased cyclic adenosine monophosphate, pentoxifylline appears to suppress, by 50% or more, endotoxin-induced mononuclear-TNF production by reducing TNF messenger RNA accumulation and by interfering with extracellular release of protein. 106 We recently demonstrated that continuous infusion of pentoxifylline to rabbits ameliorates the inflammatory changes in CSF that occur after Hib LOS exposure resulting either from direct intracisternal inoculation or from antibiotic-induced bacterial lysis. Additionally, only a slight reduction of CSF TNF concentrations is observed in animals treated with pentoxifylline. Moreover, this drug markedly attenuates meningeal inflammatory abnormalities induced by intracisternal inoculation of rabbit recom-

Volume 116 Number 5

binant IL-1/3, which suggests that this drug can interfere with later events in the inflammatory cascade, even when cytokines are present in CSFI In an experimental rat model, pentoxifyliue attenuated the Hib LOS-induced increase in BBBP up to 1 hour after 9intracisternal inoculation of endotoxin. 1~ Further studies examining its potential role in decreasing other inflammatory abnormalities, such as brain edema and intracranial pressure, and in preventing brain damage are needed before pentoxifylline can have clinical applicability. Antileukocyte CD18 receptor antibodies. The CD18 receptors are a family of adhesion-promoting proteins located on the membrane of leukocytes that appear essential for endothelium attachment and migration into extravascular tissues. For example, administration of antiCD18 monoclonal antibodies to animals blocks emigration of leukocytes into peripheral sites in response to chemotactic stimuli,s7 Tuomanem et al. 7~ demonstrated recently that intravascular injection of anti-CD 18 monoclonal antibodies blocks effectively the development of leukocytosis in the CSF of rabbits challenged intracisternally with living bacteria, endotoxin, or bacterial cell wall. This effect is associated with protection from blood-brain barrier injury, reduction of the inflammatory response during ampicillin-induced bacterial killing, prevention of brain edema, and increase in survival rates. The densities of bacteria in CSF and the rate of bacteria killing from ampicillin therapy were unaffected by the administration of the monoclonal antibody. Further studies are warranted to evaluate the efficacy and safety of this promising therapeutic approach. Corticosteroids. Corticosteroids, because of their potent antiphlogistic properties, have been used for the treatment of inflammatory diseases. Experimental bacterial meningitis in rabbits provides an excellent model to assess the efficacy and mechanisms of action of corticosteroid treatment. In experimental pneumococcal meningitis, Tauber and Sande 8~ showed that DXM therapy alone reduces intracranial hypertension, brain water content, and CSF lactate concentrations, whereas methylprednisolone affects only the development of brain edema. Additionally, Kadurugamura et al., 1~ using the same model, demonstrated that DXM lowers CSF PGE2 concentrations and decreases the leakage of proteins from serum into CSF. In the Hib meningitis model, Syrogiannopoulos et al. 13 reported that DXM alone or in combination with ceftriaxone reduces brain edema, CSF pressure, and lactate concentrations. Administration of DXM 1 hour before or simultaneously with Hib LOS significantly reduces T N F activity and the indexes of meningea~ inflammation. 27 Additionally, DXM therapy given simultaneously with or before antibiotic treatment of rabbits with Hib meningitis reduces the

Molecular pathophysiotogy of bacterial meningitis

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inflammatory response resulting from release of bacteriafree endotoxin into CSF from dying organisms. 27 This action is also associated with significant reduction in CSF T N F activity. Treatment with DXM also decreases CSF pleocytosis induced by intracisternal inoculation of rabbit recombinant IL-1.1~ In vitro studies have demonstrated that DXM inhibits production of T N F if administered to maerophages before endotoxin induction, both by diminishing the quantity of T N F messenger R N A and by preventing its translation. 1~ Once induction has occurred, DXM is incapable of regulating T N F biosynthesis. DXM also interferes with the production of IL-1 from human monocytes at different levels.110, 1H tt markedly decreases IL-1 messenger R N A by inhibiting the transcription of the IL-1 gene and by selectively decreasing the stability of IL-1 messenger RNA. In addition, DXM appears to inhibit translation of the IL-1 precursor and release of IL-1 into the extracellular fluid. Most recently, we have found that DXM profoundly inhibits transcription of IL-1 messenger R N A and biosynthesis of IL- 1[3 by astrocytoma cell lines after endotoxin challenge, regardless of when DXM was added to the cells in relation to endotoxin administration (Dr, Perry Nisen: personal communication, Jan. 16, 1990). Another mechanism of the antiinflammatory action in DXM is inhibition of phospholipase A2 activity, thereby decreasing the production of prostanoids and leukotrienes. 59 This action of DXM is probably mediated by stimulation of a group of proteins collectively termed lipocortins, 112 although its inhibitory effect on IL-1 and T N F could also account for these effects. Additionally, by acting on specific phospholipase A2, DXM may also interfere with the production of P A F by many different cells.58 Steroids have been evaluated in both uncontrolled and controlled clinical trials in human subjects with bacterial meningitis. Although contradictory results were found in initial studies, ~3, ~14 the use of methylprednisolone rather than DXM, the timing of administration, the relatively low dosage of DXM, and the inappropriate study designs and random selection of patients could expalin the differences. On the basis of results from three prospective, placebo-controlled, double-blind clinical trials with 260 infants and children,115, 116 DXM treatment is associated with significantly decreased CSF concentrations of lactate and protein and increased glucose concentrations after 24 hours of therapy, shorter duration of fever, and a lower incidence of bilateral moderate or greater hearing loss, compared with results in placebo-treated patients. Improvement in the outcome of infection in the DXM-treated children appears to be significant only for those with mild or mild to moderate disease as assessed at the time of diagnosis, compared with results in more severely affected infants. According to

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The Journal of Pediatrics May 1990

Table. C o n c e n t r a t i o n s of I L - 1 3 a n d TNFot in C S F of infants a n d children with bacterial meningitis: d e x a m e t h a s o n e and placebo groups CSF concentrations at diagnosis (No. of patients) Cytokine

IL-lfl Mean • SD (pg/ml)* Median No. (%) with detectable activity1" TNFa Mean • SD (pg/ml)* Median No. (%) with detectable activity1"

CSF concentrations '18-30 hr later (No. of patients)

Dexamethasone

Placebo

Dexamethasone

Placebo

828 • 1206 (46) 478 41 (89)

1040 • 1363 (56) 569 56 (100)

28 • 74 (46) 0 13 (28)

225 • 442 (55) 43 54 (98)

841 • 3014 (47) 12 30 (64)

744 • 2974 (59) 45 49 (83)

12 • 22 (47) 0 20 (43)

28 • 85 (59) 10 33 (56)

p

0.002