Role of intracellular chloride in the reversible activation of neutrophil beta 2 integrins: a lesson from TNF stimulation.

The process of beta(2) integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-dependent neutrophilic polymorphonuclear leukocytes' (PMN) responses to TNF. Our previous observation, showing that a marked decrease of the high basal Cl(-) content (Cl(-)(i)) is an essential step in the TNF-induced activation of PMN, stimulated this study, which investigates the role of alterations of Cl(-)(i) in the activation of beta(2) integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of beta(2) integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin alpha subunits, and epitope CBRM1/5, localized to the I domain on the alpha-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitopes are dependent on a drop in Cl(-)(i) because 1) inhibition of Cl(-)(i) decrease is invariably accompanied by inhibition of beta(2) integrin activation, 2) Cl(-)(i) decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl(-)-free buffers, activates beta(2) integrins, and 3) restoration of the original Cl(-)(i) levels is accompanied by deactivation of beta(2) integrins. We also show that Cl(-)(i) decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pH(i) does not seem to be relevant for beta(2) integrin activation. The results of our study emphasize the role of Cl(-) as a new PMN "second messenger."

TNF-Induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase.

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.

Triggering of chloride ion efflux from human neutrophils as a novel function of leukocyte beta 2 integrins: relationship with spreading and activation of the respiratory burst.

PMN residing on immobilized fibronectin have been shown to respond to TNF with an intense and long lasting Cl- efflux that leads to a marked decrease of the unusually high basal Cl- content of these phagocytes. The finding that this Cl- efflux depends, at least in part, on beta2 integrin engagement stimulated the present investigation, which addresses the question as to whether beta2 integrins per se, in the absence of PMN agonists, are able to generate signals triggering Cl- efflux. We induced beta2 integrin cross-linking by plating PMN onto surface-bound mAbs directed against either the common beta-chain (CD18) or the individual alpha-chains (CD11a, CD11b, CD11c) of LFA-1, CR3, and gp150/95. Anti-CD18 mAbs triggered a marked release of Cl- ions, which was accompanied by spreading and activation of the respiratory burst. Cross-linking of gp150/95 and LFA-1 generated the most powerful signals for the activation of Cl- efflux. The results of three independent experimental approaches, i.e., kinetic studies, use of Cl- transport inhibitors, and modulation of Cl- efflux with different amounts of anti-beta2 integrin mAbs, indicated that Cl- efflux regulates both spreading and respiratory burst triggered by beta2 integrin cross-linking. Cl- efflux appears to be independent on either alterations of [Ca2+]i or changes in the plasma membrane potential and shows sensitivity to a raise in pHi. This study uncovers a new signaling ability of beta2 integrins and contributes to highlight the role of Cl- efflux in the outside-in signal transduction pathway regulating adherence-dependent PMN responses.

Role of the 75-kDa TNF receptor in TNF-induced activation of neutrophil respiratory burst.

The exclusive role of the 55-kDa TNF receptor (TNF-R55) as the signaling receptor in TNF-induced activation of respiratory burst by human polymorphonuclear leukocytes residing on biologic surfaces has been inferred from results obtained with receptor-specific monoclonal and polyclonal Abs. In this work, we confirm this assumption by a more direct approach, i.e., by using receptor-specific TNF mutants (p55TNF and p75TNF) and, as a novel contribution, we show that cooperation of the 75-kDa TNF receptor (TNF-R75) is required for a full blown response to the cytokine. This conclusion stems from three sets of data: 1) none of the TNF-R55-specific agonists used, i.e., mAbs or p55TNF, induced a respiratory burst comparable with that induced by TNF; 2) selective down-modulation of TNF-R75 resulted in a diminished response to TNF but not to TNF-R55-specific agonists or to the chemotactic peptide FMLP; and 3) mAbs that either block or stabilize binding of TNF to TNF-R75 inhibited the response to the cytokine, suggesting that cooperation requires not only TNF binding to the receptor but also an appropriate dissociability from it. The inhibitory effect of the Abs increased as the cytokine concentrations decreased, indicating that cooperation by TNF-R75 becomes more relevant at low TNF doses. Such a cooperation does not seem to rely on the activation of a TNF-R75-linked signaling pathway independent of TNF-R55, since the response to p55TNF and p75TNF given in combination was not higher than the response to p55TNF alone. The possible mechanisms of cooperation are discussed.

[Crisis of medicine and information]. / La crisi della medicina e l'informazione.

Western medicine is experiencing a period of crisis, which started in the middle of the twentieth century. Such crisis comes from three different directions: the lack of confidence in the capacity of physicians to solve medical problems; the difficulties in health system management, due to the need to find an equilibrium between costs and benefits; the physicians increasing specialization. Correct information and divulgation, together with evidence based medicine, represent possible ways to ameliorate this situation.

Biochemical and molecular characterization of hereditary myeloperoxidase deficiency.

Hereditary myeloperoxidase (MPO) deficiency is a neutrophil disorder characterized by the lack of peroxidase activity. Cytochemical, biochemical, spectroscopic, immunocytochemical, and genetic studies were carried out on a 5-year-old MPO-deficient subject and on her parents. The father was also MPO-deficient, whereas the mother had 24% of normal MPO activity. Although the typical absorption spectrum of MPO was absent in both the father and daughter, the father's neutrophils, and not those of the daughter, contained material antigenically related to MPO. In the MPO gene of the father, two mutations were found, each located in a different allele: a T --> C transition, causing the nonconservative replacement M251T and a 14-base deletion within exon 9. The M251T substitution occurred in the carboxy-terminal region of the light chain that is included in the heme pocket. The daughter inherited the 14-base deletion from her father. The study of the MPO mRNAs present in liquid cultures of granulocyte precursors surprisingly showed that the same genetic defect, ie, the 14-base deletion, seemed to exhibit different mRNA phenotypes in the father and the daughter. In fact, mRNA derived from the 14-base-deleted allele was not found in the father and an aberrantly spliced MPO mRNA with a 77-base deletion of exon 9, which includes the 14-base deletion and leads to the generation of a premature stop codon, was found in the daughter. The possibility that Delta77 mRNA could derive from other mutations linked to the Delta14 allele was dismissed because no sequence differences were found in the region (exons and exon-intron junctions). Our data indicate that the alteration of the mRNA context caused by the 14-base deletion provide a basis for the 77-base deletion in the mRNA processing. Since the granulocyte precursors from the liquid cultures of the father were more differentiated than those from the daughter, the observed different behavior of the 14-base-deleted allele in the father and daughter may be the result of a differentiation-stage dependent control of altered spliced mRNA, which may be tolerated during the early stages of differentiation but degraded at later stages. In the liquid cultures of the daughter's cells, in addition to the mRNA with the 77-base deletion, a mRNA with the wild type sequence was also found. This mRNA was inherited from the mother, since no mutations were found in her MPO cDNA and MPO gene. The MPO defect might be caused by a regulatory mutation that induces the MPO gene switch off at an early stage of granulocyte differentiation.

Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils stimulated by tumor necrosis factor-alpha (TNF) on biologic surfaces.

Chloride ion efflux is an early event occurring after exposure of neutrophilic polymorphonuclear leukocytes (PMN) in suspension to several agonists, including cytokines such as tumor necrosis factor-alpha (TNF) and granulocyte/macrophage-colony stimulating factor (Shimizu, Y., R.H. Daniels, M.A. Elmore, M.J. Finnen, M.E. Hill, and J.M. Lackie. 1993. Biochem. Pharmacol. 9:1743-1751). We have studied TNF-induced Cl- movements in PMN residing on fibronectin (FN) (FN-PMN) and their relationships to adherence, spreading, and activation of the respiratory burst. Occupancy of the TNF-R55 and engagement of beta 2 integrins cosignaled for an early, marked, and prolonged Cl- efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between Cl- efflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced Cl- efflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of Cl- efflux, such as ethacrynic acid. Moreover, Cl- efflux induced by means other than TNF treatment, i.e., by using Cl(-)-free media, was followed by increased adherence, spreading, and metabolic activation, thus mimicking TNF effects. These studies provide the first evidence that a drastic decrease of Cl-i in FN-PMN may represent an essential step in the cascade of events leading to activation of proadhesive molecules, reorganization of the cytoskeleton network, and assembly of the O2(-)-forming NADPH oxidase.

Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family.

F4/80 is a monoclonal antibody that recognizes a murine macrophage-restricted cell surface glycoprotein and has been extensively used to characterize macrophage populations in a wide range of immunological studies. Apart from the tightly regulated pattern of expression of the F4/80 antigen, little is known about its possible role in macrophage differentiation and function. We have sought to characterize the molecule at the molecular level, through the isolation of cDNA clones, and now describe the sequence of the F4/80 protein. The primary amino acid sequence demonstrates homology to two protein superfamilies. The NH2-terminal region consists of seven epidermal growth factor-like domains, separated by approximately 300 amino acids from a COOH-terminal region that shows homology to members of the seven transmembrane-spanning family of hormone receptors. The potential role of these distinct domains is discussed with respect to the possible function of the F4/80 molecule.

Human eosinophil peroxidase enhances tumor necrosis factor and hydrogen peroxide release by human monocyte-derived macrophages.

Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth. In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte-derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12-myristate 13-acetate-triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear-like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H2O2-degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myeloperoxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO-induced effects differed in several aspects from the effects of lipopolysaccaride and interferon-gamma, two well-known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.

Hereditary eosinophil peroxidase deficiency: immunochemical and spectroscopic studies and evidence for a compound heterozygosity of the defect.

Hereditary eosinophil peroxidase (EPO; EC 1.11.1.7) deficiency is a rare abnormality of eosinophil granulocytes characterized by decreased or absent peroxidase activity and decreased volume of the granule matrix. The molecular basis of the defect is not known. We report here its molecular characterization in an EPO-deficient subject and his family members. The EPO-deficient eosinophils contained EPO-related material as determined immunochemically using either monoclonal or polyclonal anti-EPO antibodies but had no spectroscopic evidence of EPO. Eosinophil precursors from the EPO-deficient subject contained normally sized EPO mRNA, which was reverse transcribed into the corresponding cDNA clones encompassing the whole gene. Sequencing of these clones disclosed two mutations, a G-->A transition causing a nonconservative replacement of an arginine residue with a histidine and an insertion causing a shift in the reading frame with the appearance of a premature stop codon. The two mutations were located on different chromosomes indicating a compound heterozygosity for the defect. Both the son and the daughter of the proband inherited the G-->A transition, and their eosinophils contained a peroxidase activity intermediate between that of control subjects and the proband, suggesting that the transition is a deficiency-causing mutation. Eosinophil precursors from the EPO-deficient subject were found to actively synthesize an EPO that was apparently normal in terms of cytochemical reaction for peroxidase and immunoreactivity with monoclonal and polyclonal anti-EPO antibodies, but spectroscopically abnormal. The cytochemical reaction for peroxidase tended to decrease or disappear in the eosinophil precursors of the EPO-deficient subject but not of a normal subject as differentiation went on, suggesting that the Arg-->His substitution causes the production of an unstable EPO that undergoes progressive degradation as the cells mature.

Evidence that tumor necrosis factor alpha (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor.

Polymorphonuclear leukocytes (PMN) residing on biologic surfaces respond with a vigorous respiratory burst when exposed to tumor necrosis factor alpha (TNF). PMN possess both the p55 and the p75 TNF receptors, but their role in the elicitation of the respiratory burst is not known. We addressed this problem by studying the effect of monoclonal antibodies (MoAbs) directed against the p55 TNF receptor (MoAb H398 and MoAb htr-9) and the p75 TNF receptor (MoAb utr-1) on TNF-induced production of O2- by PMN residing on fibronectin-coated surfaces. Neither the anti-p55 nor the anti-p75 MoAbs affected TNF-induced O2- production despite their known ability to competitively inhibit TNF binding to the corresponding receptor. Experiments with the antibodies alone showed that the anti-p55 MoAbs directly triggered PMN O2- production, whereas no response was elicited by the anti-p75 MoAb. PMN unresponsiveness to the anti-p75 MoAb could not be ascribed to low expression of p75 receptor, because binding of the anti-p75 MoAb utr-1 to PMN was, indeed, even higher than binding of the anti-p55 MoAb htr-9. The agonistic activity of the anti-p55 MoAbs was comparable with that of TNF and was not or only minimally modified by the simultaneous presence of TNF. Triggering of the respiratory burst by TNF was completely prevented by Fab fragments of the anti-p55 MoAb H398. Moreover, the monovalent Fab fragments, which lacked any stimulatory effect on PMN O2- production, acquired strong agonistic activity on cross-linking with anti Fab antibodies, suggesting that the ability of the anti-p55 antibodies to stimulate PMN O2- production depends on their ability to cross-link the TNF receptors. The agonistic effect of the anti-p55 MoAbs was only observed with cells residing on fibronectin-coated surfaces and not with cells in suspension, and in terms of kinetics, dependence on beta 2 integrin-mediated adherence, microfilament integrity, and sensitivity to elevations of intracellular levels of cAMP, it was virtually indistinguishable from the agonistic effect of TNF. Taken together, these results suggest that the p55 receptor is responsible for TNF-induced triggering of the respiratory burst of PMN residing on biologic surfaces.

Effect of a standardized liver and spleen fraction of peptides on the differentiation of human monocyte-derived macrophages.

The effect of Factor AF2 (AF2), a standardized fraction of peptides with a molecular weight of < 10,000 Dalton obtained from livers and spleens of newborn lambs, on the differentiation of human monocyte-derived macrophages was studied, in view of the central role played by these cells in inflammation and tumor cytotoxicity. The results show that the drug 1. increases the cell density of cultures, 2. favours the morphologic differentiation of monocytes into macrophages, and 3. increases the macrophages phagocytic capacity. The first two effects are observed when monocytes are cultured in 1% serum but not in 10% serum while the enhancement of phagocytic activity is detected at both serum concentrations.

Effect of a protein-free dialysate from calf blood on human monocyte differentiation in vitro.

Solcoseryl is a protein-free, standardized dialysate/ultrafiltrate derived from calf blood, which has been shown to improve situations of impaired healing in both experimental animals and man. Its activity seems to be multifactorial although the precise cellular and molecular mechanisms contributing to its effect have not been fully elucidated. Since monocyte-derived macrophages play a central role in inflammation and particularly in wound healing and tissue remodelling, the effect of the dialysate on human monocytes cultured in vitro for 10 days in the presence of human serum was studied. The results show that the drug, at concentrations ranging from 0.2 to 2%, increases the cell density of the cultures and the cell protein content, and favours the differentiation of monocytes into macrophages when 1% serum concentration is used in the culture medium. These effects are no more apparent when the serum concentration was raised to 10%. These data suggest that the drug may substitute, at least in part, for serum in monocyte-macrophage cultures. The observed effects give a sound basis for at least a partial explanation of the therapeutic effects of the drug, particularly at sites where the supply of serum-derived factors is limited.

Potentiation of human polymorphonuclear leukocytes respiratory burst and phagocytosis by a standardized liver and spleen fraction of peptides.

The effect of Factor AF2 (AF2), a xenogeneic fraction of peptides with a molecular weight of < 10,000 Dalton obtained from livers and spleens of newborn lambs, on the oxygen consumption and the phagocytic activity of human polymorphonuclear leukocytes (PMN) was studied. AF2 increased the oxygen uptake of PMN exposed both to serum-treated zymosan (STZ), a phagocytosable stimulus, and phorbol-myristate-acetate (PMA), a soluble stimulus. The potentiating effect of the drug was dose-dependent and more pronounced when suboptimal amounts of either stimulus were used. The phagocytic activity of PMN, as measured by the rate of mineral oil particles ingestion, was also increased by AF2 in a dose-dependent manner. These results suggest that the drug may influence PMN behaviour in at least two ways: 1. by increasing the rate of phagocytosis, and 2. by potentiating the respiratory burst induced by soluble and particulate stimuli. The results are discussed in relation to the beneficial effects of AF2 in cancer patients under chemotherapy or radiation treatment.

A simple and rapid method for isolation of eosinophilic granulocytes from human blood.

A simple and rapid method for the purification of morphologically and functionally intact eosinophils from human blood of both normal and eosinophilic subjects is described. The method is based on a single centrifugation of total blood leukocytes suspended in Percoll with specific gravity 1.0853 g/ml, after erythrocyte removal by dextran sedimentation. The peculiarity of this isolation technique is the maintenance of strictly physiological values of pH and osmolality throughout the entire procedure. Moreover, the cells are not subjected to measures aimed at changing the physical properties of either neutrophils or eosinophils. Because of such characteristics, this isolation method could be usefully exploited for comparative studies of normal and eosinophilic normodense eosinophils and of neutrophils and eosinophils from the same noneosinophilic subject.

Simultaneous assay for oxidative metabolism and adhesion of human neutrophils: evidence for correlations and dissociations of the two responses.

An assay method for the simultaneous evaluation of the oxidative metabolism and adherence of human neutrophils is described, together with certain specific applications. Incubations were performed in serum-coated microtiter plates, where oxidative metabolism was measured as O2- release and, after washing out the nonadherent cells, the adhesion was measured as activity of acid phosphatase. Three agonists tested in this system--opsonized zymosan, concanavalin A, and N-formyl-methionyl-leucyl-phenylalanine--induced both activation of O2- release and cell adhesion, but the two functions had time course and dose dependence patterns that varied depending on the stimulant. Particularly with concanavalin A, O2- release and adhesion response were markedly dissociated; this lectin at low doses increased neutrophil adherence without triggering any O2- production, whereas at high doses it increased both O2- production and adherence. Anti-integrin monoclonal antibodies did not affect adhesion induced by low-dose concanavalin A but inhibited the adhesion induced by the other tested agonists. Adhesion and O2- production were also found to be differentially affected by the NADPH oxidase inhibitor diphenylene iodonium, the sulfhydryl reagent N-ethylmaleimide and the A2 agonist adenosine, indicating that these neutrophil responses have various transductional pathways that also depend on the type of stimulus.

Eosinophil activation on biologic surfaces. Production of O2- in response to physiologic soluble stimuli is differentially modulated by extracellular matrix components and endothelial cells.

Production of O2- in response to FMLP, TNF, IFN-gamma, platelet activating factor, LPS, substance P, and PMA by human eosinophils in suspension and in contact with polystyrene ELISA plastic (PL) or biologic surfaces was studied. Monolayers of human endothelial cells (HEC) or PL coated with FCS, fibronectin, laminin, collagen types I and IV, fibrinogen, or fibrin were used as biologic surfaces. Only PMA and FMLP stimulated O2- generation by eosinophils in suspension. Eosinophils residing on HEC monolayers, either untreated or treated with LPS, were unresponsive to all stimuli except PMA. PMA induced O2- generation by eosinophils on all surfaces; FMLP on all surfaces but HEC monolayers; TNF and platelet-activating factor only on PL, fibrinogen, and fibrin; LPS and substance P only on PL. PMA was equally effective on eosinophils on surfaces and in suspension, whereas the effect of FMLP was greater on eosinophils on surfaces than on eosinophils in suspension. IFN-gamma was ineffective on any of the surfaces tested. These results indicate that biologic surfaces may profoundly affect the ability of eosinophils to respond with a respiratory burst to physiologically relevant soluble stimuli, the effect varying according to the nature of both the stimulus and the surface. Since the respiratory burst generates products of oxygen reduction that are toxic to several tissue components, it follows that biologic surfaces may modulate eosinophil-induced tissue injury.

Effect of biological surfaces on neutrophil O2- production and its relationship to the CD11b/CD18 integrin-dependent adherence.

The production of O2- in response to LPS, PAF, FMLP, TNF and PMA by human neutrophils in suspension and residing on surfaces coated with fetal calf serum (FCS), fibronectin (FN), laminin (LM), collagen types I and IV (CI and CIV), fibrinogen (FBG) or fibrin (FBN) was studied. Of the agonists used, PAF and LPS failed to induce a response in any of the above conditions; FMLP and PMA stimulated neutrophils to produce similar amounts of O2- either in suspension or on biological surfaces; TNF induced O2- production only by cells residing on FN, FBG and FBN. These results indicate that production of oxygen-derived free radicals by neutrophils depends on the type of agonist and the nature of the surface they interact with. The relationship between the respiratory burst and adherence was studied by measuring O2- release and adherence of neutrophils residing on FN, LM, CIV, and FBG, in the absence and in the presence of the monoclonal antibody 60.3 that recognizes the common beta-chain of CD11/CD18 integrins. FMLP, PMA and TNF increased neutrophil adherence on all these surfaces except CIV. The monoclonal antibody markedly inhibited the FMLP and PMA-induced adherence but had no effect on the O2- release elicited by these two agonists. In contrast, the monoclonal antibody inhibited both the increased adherence and O2- release induced by TNF on FN and FBG. The TNF-induced increase in adherence to LM, that was not accompanied by an increase in O2- release, was also inhibited by the monoclonal antibody. We conclude that the respiratory burst of neutrophils residing on surfaces is not necessarily correlated with adherence.

Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain.

We have examined the distribution of microglia in the normal adult mouse brain using immunocytochemical detection of the macrophage specific plasma membrane glycoprotein F4/80. We were interested to learn whether the distribution of microglia in the adult brain is related to regional variation in the magnitude of cell death during development and resulting monocyte recruitment, or whether the adult distribution is influenced by other local microenvironmental cues. We further investigated the possibility that microglia are sensitive to their microenvironment by studying their morphology in different brain regions. Microglia are present in large numbers in all major divisions of the brain but are not uniformly distributed. There is a more than five-fold variation in the density of immunostained microglial processes between different regions. More microglia are found in gray matter than white. Particularly, densely populated areas include the hippocampus, olfactory telencephalon, basal ganglia and substantia nigra. In comparison, the less densely populated areas include fibre tracts, cerebellum and much of the brainstem. The cerebral cortex, thalamus and hypothalamus have average cell densities. There was no simple relationship between the amount of developmental cell death and the adult distribution of microglia. An estimate of the total number of microglia in the adult mouse brain, 3.5 x 10(6), is comparable to that found in the liver on a weight for weight basis. However, microglia possess up to twice the surface area of membrane of Kupffer cells, the large resident macrophages of the liver. The proportion of cells that were microglia varied from 5% in the cortex and corpus callosum, to 12% in the substantia nigra. Microglia vary in morphology depending on their location. They were broadly classified into three categories. Compact cells are rounded cells, sometimes with one or two short thick limbs, bearing short processes ("bristles"). They resemble Kupffer cells of the liver and are found exclusively in sites lacking a blood-brain barrier. Longitudinally branched cells are found in fibre tracts and possess several long processes which are usually aligned parallel to, or more occasionally perpendicular to, the longitudinal axis of the nerve fibres. Radially branched cells are found throughout the neuropil. They can be extremely elaborate and there is wide variation in the length and complexity of branching of the processes. There was no evidence of monocyte-like cells in the adult CNS. The systematic variation in microglial morphology provides further evidence that these cells are sensitive to their microenvironment.