Christian U.A. Kloss

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.

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.

Integrin family of cell adhesion molecules in the injured brain: Regulation and cellular localization in the normal and regenerating mouse facial motor nucleus

Integrins are a large family of heterodimeric glycoproteins that play a crucial role in cell adhesion during development, inflammation, and tissue repair. In the current study, we investigated the localization of different integrin subunits in the mouse facial motor nucleus and their regulation after transection of the facial nerve. In the normal mouse brain, there was clear immunoreactivity for α5‐, α6‐, and β1‐integrin subunits on blood vessel endothelia and for αM‐ and β2‐subunits on resting parenchymal microglia. Facial nerve transection led to an up‐regulation of the β1‐subunit on the axotomized neurons and an increase in the α4‐, α5‐, α6‐, β1‐, αM‐, αX‐, and β2‐subunits on the adjacent, activated microglia. Quantification of the microglial integrins revealed two different expression patterns. The subunits α5 and α6 showed a monophasic increase with a maximum at day 4, the αM‐subunit a biphasic regulation, with an early peak at day 1 and an elevated plateau between day 14 and 42. At day 14, there was also an influx of lymphocytes immunoreactive for the α4β1‐ and αLβ2‐integrins, which aggregated at sites of neural debris and phagocytotic microglia. This finding was accompanied by a significant increase of the α5β1‐integrin on blood vessel endothelia. In summary, facial axotomy is followed by a strong and cell‐type–specific expression of integrins on the affected neurons and on surrounding microglia, lymphocytes, and vascular endothelia. The presence of several, strikingly different temporal patterns suggests a selective involvement of these molecules in the different adhesive events during regeneration in the central nervous system. J. Comp. Neurol. 411:162–178, 1999.

Microglia and the early phase of immune surveillance in the axotomized facial motor nucleus: Impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage‐colony stimulating factor‐deficient mice

Activation of microglia is among the first cellular changes in the injured CNS. However, little is known about their specific contribution to secondary damage or repair processes in neighboring neurons and nonneuronal cells or to the immune surveillance of the damaged tissue. Animal models with defective microglial response such as osteopetrosis provide an approach to explore these effects. Osteopetrosis (op) is an autosomal recessive mutation with a complete deficiency of the macrophage‐colony stimulating factor (MCSF; CSF‐1), an important mitogen for brain microglia. In the current study we examined the effects of this MCSF deficiency on the microglial reaction and the overall cellular response to nerve injury in the mouse axotomized facial motor nucleus. In the brain, MCSF receptor immunoreactivity was found only on microglia and was strongly up‐regulated following injury. MCSF deficiency led to a failure of microglia to show a normal increase in early activation markers (thrombospondin, MCSF receptor, αMβ2‐ and α5β1‐integrins), to spread on the surface of axotomized motoneurons, and to proliferate after injury. Early recruitment of CD3+ T‐lymphocytes to the facial nucleus 24 hours after injury was reduced by 60%. In contrast, the neuronal and astrocyte response was not affected. There was a normal increase in the neuropeptides calcitonin gene‐related peptide and galanin, neuronal c‐JUN, and NADPH‐diaphorase and a decrease in choline acetyltransferase and acetylcholinesterase. Astrocyte glial fibrillary acidic protein immunoreactivity also showed a normal increase. There was a normal influx of macrophages and granulocytes into the injured facial nerve. Synaptic stripping, neuronal survival, and speed of axonal regeneration were also not affected. The current results show a strong, selective effect of MCSF on the early activation of microglia and, indirectly, on lymphocyte recruitment. This early phase of microglial activation appears not to be involved in the process of repair following peripheral nerve injury. However, it is important in the initiation of inflammatory changes in the brain and in the interaction with the immune system. J. Comp. Neurol. 436:182–201, 2001.

Proliferation of ramified microglia on an astrocyte monolayer: Characterization of stimulatory and inhibitory cytokines

Proliferation of ramified microglia is a common phenomenon in brain pathology, but little is known about how this is regulated. In the current study, we examined the effect of different cytokines on the proliferation of ramified microglia in vitro using a combination of autoradiography for [3H]‐thymidine and immunocytochemical techniques.

Regulation of MSCF receptors on microglia in the normal and injured mouse central nervous system: A quantitative immunofluorescence study using confocal laser microscopy

The macrophage colony‐stimulating factor (MCSF) is a 40–76‐kD glycoprotein that plays an important role in the activation and proliferation of microglia both in vitro and in injured neural tissue. Here, we examined the regulation of MCSF receptor (MCSFR) and MCSF in the normal and injured mouse central nervous system (CNS) by using confocal laser microscopy, quantitative immunofluorescence, and reverse transcriptase–polymerase chain reaction (RT‐PCR) techniques.

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.

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.

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 αMβ2‐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 μg/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 μg/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. J. Neurosci. Res. 64:508–522, 2001.

Lymphocyte infiltration in the injured brain: Role of proinflammatory cytokines

Studies using mouse axotomised facial motoneuron model show a strong and highly selective entry of CD3+ lymphocytes into the affected nucleus, with a maximum at Day 14, which coincides with the peak of neuronal cell death, microglial phagocytosis, and increased synthesis of interleukin‐1 beta (IL1β), tumour necrosis factor‐alpha (TNFα) and interferon‐gamma (IFNγ). We explored the possible involvement of these cytokines during the main phase of lymphocyte recruitment into the axotomised facial motor nucleus 7–21 days after nerve cut using mice homozygously deficient for IL1 receptor type 1 (IL1R1−/−), TNF receptor type 1 (TNFR1−/−), type 2 (TNFR2−/−) and type 1 and 2 (TNFR1&2−/−), IFNγ receptor type 1 (IFNγR1−/−), and the appropriate controls for the genetic background. Transgenic deletion of IL1R1 led to a 54% decrease and that of TNFR2 to a 44% reduction in the number of CD3+ T‐cells in the axotomised facial motor nucleus, with a similar relative decrease at Day 7, 14, and 21. Deletion of TNFR1 or IFNγR1 had no significant effect. Deletion of both TNFR1 and 2 (TNFR1&2−/−) caused a somewhat stronger, 63% decrease than did TNFR2 deletion alone, but this could be due to an almost complete inhibition of neuronal cell death. No mutations seemed to inhibit aggregation of CD3+ T‐cells around glial nodules consisting of Ca‐ion binding adaptor protein‐1 (IBA1)+ phagocytotic microglia and neuronal debris. Altogether, the current data show the importance of IL1R1 and TNFR2 as the key players during the main phase of lymphocyte recruitment to the damaged part of the central nervous system.