Major histocompatibility complex (MHC2+) perivascular macrophages in the axotomized facial motor nucleus are regulated by receptors for interferon-gamma (IFNgamma) and tumor necrosis factor (TNF).

The major histocompatibility complex (MHC) glycoproteins, MHC1 and MHC2, play a key role in the presentation of antigen and the development of the immune response. In the current study we examined the regulation of the MHC2 in the mouse brain after facial axotomy. The normal facial motor nucleus showed very few slender and elongated MHC2+ cells. Transection of the facial nerve led to a gradual but strong upregulation in the number of MHC2+ cells, beginning at day 2 and reaching a maximum 14 days after axotomy, correlated with the induction of mRNA for tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and interferon-gamma (IFNgamma) and a peak in neuronal cell death. In almost all cases, MHC2 immunoreactivity was restricted to perivascular macrophages that colocalized with vascular basement membrane laminin and macrophage IBA1-immunoreactivity, with no immunoreactivity on phagocytic microglia, astrocytes or invading T-cells. Heterologous transplantation and systemic injection of endotoxin or IFNgamma did not affect this perivascular MHC2 immunoreactivity, and transgenic deletion of the IL1 receptor type I, or TNF receptor type 1, also had no effect. However, the deletion of IFNgamma receptor subunit 1 caused a significant increase, and that of TNF receptor type 2 a strong reduction in the number of MHC2+ macrophages, pointing to a counter-regulatory role of IFNgamma and TNFalpha in the immune surveillance of the injured nervous system.

Microglial major histocompatibility complex glycoprotein-1 in the axotomized facial motor nucleus: regulation and role of tumor necrosis factor receptors 1 and 2.

Presentation of antigen is key to the development of the immune response, mediated by association of antigen with major histocompatibility complex glycoproteins abbreviated as MHC1 and MHC2. In the current study, we examined the regulation of MHC1 in the brain after facial axotomy. The normal facial motor nucleus showed no immunoreactivity for MHC1 (MHC1-IR). Transection of the facial nerve led to a strong and selective up-regulation of MHC1-IR on the microglia in the affected nucleus, beginning at day 2 and reaching a maximum 14 days after axotomy, coinciding with a peak influx of the T lymphocytes that express CD8, the lymphocyte coreceptor for MHC1. Specificity of the MHC1 staining was confirmed in beta2-microglobulin-deficient mice, which lack normal cell surface MHC1-IR. MHC1-IR was particularly strong on phagocytic microglia, induced by delayed neuronal cell death, and correlated with the induction of mRNA for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and interferon-gamma and the influx of T lymphocytes. Mice with severe combined immunodeficiency (scid), lacking T and B cells, showed an increase in the number of MHC1-positive nodules but no significant effect on overall MHC1-IR. Transgenic deletion of the IL1 receptor type I, or the interferon-gamma receptor type 1 subunit, did not affect the microglial MHC1-IR. However, a combined deletion of TNF receptors 1 and 2 (TNFR1&2-KO) led to a decrease in microglial MHC1-IR and to a striking absence of the phagocytic microglial nodules. Deletion of TNFR2 (p75) did not have an effect; deletion of TNFR1 (p55) reduced the diffuse microglial staining for MHC1-IR but did not abolish the MHC1(+) microglial nodules. In summary, neural injury leads to the induction of MHC1-IR on the activated, phagocytic microglia. This induction of MHC1 precedes the interaction with the immune system, at least in the facial motor nucleus model. Finally, the impaired induction of these molecules, up to now, only in the TNFR-deficient mice underscores the central role of TNF in the immune activation of the injured nervous system.

B7.2 on activated and phagocytic microglia in the facial axotomy model: regulation by interleukin-1 receptor type 1, tumor necrosis factor receptors 1 and 2 and endotoxin.

Co-stimulatory factors are involved in different forms of brain pathology and play an important role in the activation of T-cells. In the current study, we explored the regulation of B7.2, a prominent member of the B7 family of costimulatory factors, in the facial motor nucleus (FMN) following facial axotomy and systemic application of lipopolysaccharide (LPS, endotoxin) using light and electron immunohistochemistry and cytokine-receptor-deficient mice. Facial axotomy led to a gradual increase of B7.2 immunoreactivity (IR) on microglial cell surface; similar effects were also observed following application of LPS, but both effects were not additive, suggesting overlapping or saturated signaling pathways. Some B7.2-IR was already present on activated microglia surrounding injured neurons at days 1-4 after injury, but became particularly intense during neuronal cell death, peaking at day 14. Previous studies revealed that these late microglial changes are accompanied by a strong increase in the expression of proinflammatory cytokines such as interleukin-1 beta (IL1beta) tumor necrosis factor-alpha (TNFalpha) and interferon gamma (IFNgamma) [J. Neurosci. 18 (1998a) 5804]. Here, deletion of the receptors for these cytokines-IL1R1, TNFR1 or TNFR2, but not IFNgammaR1-caused a strong and significant reduction in B7.2-IR in reactive microglial cells, compared with their wild type (WT) controls on the same genetic strain background, with a 31% decrease in IL1R1-/- , 39% in TNFR1-/- and 49% in TNFR2-/- mice. These data underscore the significance of IL1beta, TNFalpha and LPS, and their receptors, as potent inflammatory signals that regulate the cellular response in the injured brain as well as the interaction with the rapidly recruited immune system. The broad susceptibility of B7.2 regulation to a wide range of different inflammatory signals also points to its role as a sensor of molecular pathology, and a factor that plays an important accessory role in allowing and shaping the microglia/T-cell interaction in the injured central nervous system.

Systemic LPS injection leads to granulocyte influx into normal and injured brain: effects of ICAM-1 deficiency.

The lipopolysaccharide (LPS) constituents of the gram-negative bacterial wall are among the most potent activators of inflammation. In the current study, we examined the effect of subcutaneous injection of Escherichia coli LPS on leukocyte influx into the normal and injured brain using endogenous peroxidase (EP). Normal brain parenchyma does not contain granulocytes and this does not change after indirect trauma, in facial axotomy. However, systemic injection of 1 mg LPS led to a gradual appearance of EP-positive parenchymal granulocytes within 12 h, with a maximum at 1-4 days after injection. Facial axotomy (day 14) led to a further 50-300% increase in granulocyte number. Of the five mouse strains tested in the current study, four--Balb/C, FVB, C57Bl/6, and C3H/N--showed vigorous granulocyte influx (60-90 cells per 20-microm section in axotomized facial nucleus, 20-40 cells per section on the contralateral side). The influx was an order of magnitude lower in the SJL mice. The peroxidase-positive cells were immunoreactive for neutrophil antigen 7/4 and alpha M beta 2 integrin, were negative for IBA1 (monocytes) and CD3 (T cells), and could be prelabeled by subcutaneous injection with rhodamine B isothiocyanate (RITC), confirming their origin as blood-borne granulocytes. All RITC-positive cells were IBA1 negative. This influx of granulocytes was accompanied by a disruption of the blood-brain barrier to albumin and induction of the cell adhesion molecule ICAM-1 on affected blood vessels. Transgenic deletion of ICAM-1 led to a more than 50% reduction in the number of infiltrating granulocytes compared to litter-matched wild-type controls, in normal brain as well as in axotomized facial motor nucleus. In summary, systemic injection of LPS leads to invasion of granulocytes into the mouse brain and a breakdown of the blood-brain barrier to blood-borne cells and to soluble molecules. Moreover, this mechanism may play a pathogenic role in the etiology of meningitis and in severe bacterial sepsis.

In vitro model of microglial deramification: ramified microglia transform into amoeboid phagocytes following addition of brain cell membranes to microglia-astrocyte cocultures.

Changes in the morphology of ramified microglia are a common feature in brain pathology and culminate in the appearance of small, rounded, microglia-derived phagocytes in the presence of neural debris. Here, we explored the effect of adding brain cell membranes on the morphology of alphaMbeta2-integrin (CD11b/CD18, CR3) positive microglia cultured on a confluent astrocyte substrate as an in vitro model of deramification. Addition of brain membranes led to a loss of microglial ramification, with full transformation to small, rounded, macrophages at 20-40 microg/ml. Time course studies showed a rapid response, with first effects at 1-3 hours, and full transformation at 24-48 hours. Removal of cell membranes and exchange of the culture medium led to a similarly rapid process of reramification. Comparison of cell membranes from different tissues at 20 microg/ml showed strong transforming effect for the brain, more moderate for kidney and liver, and very weak for spleen and skeletal muscle. Fluorescent labeling of brain membranes revealed uptake by almost all rounded macrophages, by a subpopulation of glial fibrillary acidic protein (GFAP)-positive astrocytes, but not by ramified microglia. Phagocytosis of inert fluorobeads did not lead to a transformation into macrophages but their phagocytosis was inhibited by brain membranes, pointing to a saturable uptake mechanism. In summary, addition of brain cell membranes and their phagocytosis leads to a rapid and reversible loss of ramification. The differences in transforming activity from different tissues and the absence of effect from phagocytosed fluorobeads suggest, however, the need for a second stimulus following the phagocytosis of cell debris.

Effect of lipopolysaccharide on the morphology and integrin immunoreactivity of ramified microglia in the mouse brain and in cell culture.

Microglial cells form the first line of defense in brain infection. They are related to monocytes and macrophages and can be readily activated by cell wall components of bacteria such as lipopolysaccharides (LPS). In the present study, we explored the effect of this endotoxin in mouse on the morphology of microglia and their immunoreactivity for the integrin family of cell adhesion molecules in vitro and in vivo. Subcutaneous injection of LPS led to a dose-dependent activation of alpha M beta 2-positive microglia, with a saturating effect at 1 microg LPS in the blood-brain barrier deficient area postrema, at 10 microg in the directly adjacent tissue, and at 100 microg throughout the brainstem and cerebellum. Morphologically, this activation was characterized by the swelling of the microglial cell body, a thickening of the proximal processes, and a reduction in distal ramification. Microglial immunoreactivity for the integrins alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1, and alpha M beta 2 was strongly increased. In vitro, ramified microglia were obtained using a coculture on top of a confluent astrocyte monolayer. Two days exposure to LPS resulted in a morphological activation of the cultured cells with an increase of the integrin immunoreactivity for alpha 5 (5.7-fold), alpha 4 (3.1-fold), beta 1 (2.3-fold), and alpha M (1.5-fold), and a decrease in the alpha 6-staining intensity by 39%. Even a sublethal dose of LPS (3 mg in vivo and 500 microg/ml in vitro, respectively) did not induce the phagocyte-associated integrin alpha X beta 2 (CD11c/CD18, p150,95) and did not lead to a morphological transformation of the ramified microglia into phagocytes.

Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function.

Damage to the central nervous system (CNS) leads to cellular changes not only in the affected neurons but also in adjacent glial cells and endothelia, and frequently, to a recruitment of cells of the immune system. These cellular changes form a graded response which is a consistent feature in almost all forms of brain pathology. It appears to reflect an evolutionarily conserved program which plays an important role in the protection against infectious pathogens and the repair of the injured nervous system. Moreover, recent work in mice that are genetically deficient for different cytokines (MCSF, IL1, IL6, TNFalpha, TGFbeta1) has begun to shed light on the molecular signals that regulate this cellular response. Here we will review this work and the insights it provides about the biological function of the neuroglial activation in the injured brain.

Cytotoxic potential of proinflammatory cytokines: combined deletion of TNF receptors TNFR1 and TNFR2 prevents motoneuron cell death after facial axotomy in adult mouse.

Neural injury is known to trigger inflammatory changes, including the synthesis of proinflammatory cytokines such as interleukin-1-beta (IL1beta), tumor necrosis factor-alpha (TNFalpha), and interferon-gamma (IFNgamma) [G. Raivich, L. L. Jones, C. U. A. Kloss, A. Werner, H. Neumann, and G. W. Kreutzberg, 1998, J Neurosci, 18: 5804-5816] that may play a pivotal role in mediating the cellular response in the affected brain tissue. Here we examined the effects of transgenic deletion of receptors for these cytokines on neuronal cell loss in the adult mouse facial motor nucleus after a peripheral, facial nerve cut. Homozygous deletion of IL1 receptor 1 (IL1R1), TNF receptor 1 or 2 (TNFR1 or TNFR2), or IFNgamma receptor 1 (IFNgammaR1) alone had no effect but combined deletion of TNFR1 and TNFR2 caused a striking absence of alphaX beta2 integrin/IBA1-double-labeled, phagocytic microglial nodules in the axotomized facial motor nucleus 14 days after nerve cut. Moreover, this combined deletion also led to an almost complete prevention of cell loss by Day 29. Additional neuronal cell counts at Day 60 revealed a second phase of motoneuron cell disappearance, which did not depend on the presence of TNF receptors. However, there was still the same 22% difference in the total number of motoneurons between the wild-type and TNFR1 & 2-deficient mice, underlining the role of TNF ligands and both TNF receptors in mediating the early phase of neuronal cell loss after traumatic injury.

Interleukin-6 (IL6) and cellular response to facial nerve injury: effects on lymphocyte recruitment, early microglial activation and axonal outgrowth in IL6-deficient mice.

Nerve injury triggers numerous changes in the injured neurons and surrounding non-neuronal cells. Of particular interest are molecular signals that play a role in the overall orchestration of this multifaceted cellular response. Here we investigated the function of interleukin-6 (IL6), a multifunctional neurotrophin and cytokine rapidly expressed in the injured nervous system, using the facial axotomy model in IL6-deficient mice and wild-type controls. Transgenic deletion of IL6 caused a massive decrease in the recruitment of CD3-positive T-lymphocytes and early microglial activation during the first 4 days after injury in the axotomized facial nucleus. This was accompanied by a more moderate reduction in peripheral regeneration at day 4, lymphocyte recruitment (day 14) and enhanced perikaryal sprouting (day 14). Motoneuron cell death, phagocytosis by microglial cells and recruitment of granulocytes and macrophages into injured peripheral nerve were not affected. In summary, IL6 lead to a variety of effects on the cellular response to neural trauma. However, the particularly strong actions on lymphocytes and microglia suggest that this cytokine plays a central role in the initiation of immune surveillance in the injured central nervous system.