Fanny Reichert

Murine nucleus pulposus-derived cells secrete interleukins-1-beta, -6, and -10 and granulocyte-macrophage colony-stimulating factor in cell culture.

Study Design. Cultures established from murine disc‐derived cells were stimulated by lipopolysaccharide. The cells capacity to secrete proinflammatory cytokines and interleukin‐10 with and without lipopolysaccharide stimulation was determined using enzymelinked immunosorbent assays. Objectives. To determine the capacity of disc‐derived cells to secrete proinflammatory cytokines, and the effect of lipopolysaccharide stimulation on such secretion. Summary of Background Data. The pathophysiology of compressive radiculopathy is unclear. Inflammation is a possible explanation. Proinflammatory cytokine secretion was demonstrated in herniated nucleus pulposus. It is unknown whether these cytokines are secreted from disc‐derived cells or from infiltrating inflammatory cells in the herniated nucleus pulposus. Methods. Discs were microsurgically harvested from inbred mice and cut to allow the nucleus pulposus to establish cell culture. A study group was exposed to lipopolysaccharide stimulation. Media were harvested from the study and control groups 24 hours later. Secretion of interleukins‐1‐, ‐6, and ‐10, granulocyte‐macrophage colony‐stimulating factor, and tumor necrosis factor‐α were determined using enzyme‐linked immunosorbent assays. Results. Basal secretion of interleukins‐6 and ‐10, but no basal secretion of interleukin‐1‐, granulocyte‐macrophage colony‐stimulating factor, or tumor necrosis factor‐α was detected. Secretion of interleukin‐1‐ rose from zero to 27.69 pg/105 cells, and granulocyte‐macrophage colony‐stimulating factor secretion rose from zero to 9.77 pg/105 cells after lipopolysaccharide stimulation. A 75‐fold increase in interleukin‐6 secretion and a 150‐fold increase in interleukin‐10 secretion were detected after stimulation with lipopolysaccharide. No tumor necrosis factor‐α secretion was detectable. All result had high statistical significance (all P < 0.001). Conclusions. Cultured murine disc‐derived cells have the capacity to secrete proinflammatory cytokines and interleukin‐10 in the absence of inflammatory cells. This finding supports the hypothesis that disc‐derived cells are capable of initiating or amplifying an inflammatory process.

Interleukin 6 in Intact and Injured Mouse Peripheral Nerves

The multifunctional cytokine interleukin 6 (IL‐6) has direct growth, survival and differentiation effects on peripheral and central neurons. Furthermore, it can modulate the production by non‐neuronal cells of other cytokines and growth factors, and thereby affect nerve cells indirectly. We have studied IL‐6 expression and production in intact and injured peripheral nerves of C57/BL/6NHSD mice, which display the normal rapid progression of Wallerian degeneration. The IL‐6 mRNA was detected in nerves degenerating in vitro or in vivo, but not in intact nerves. In vitro‐ and in vivo‐degenerating nerve segments and neuroma nerve segments synthesized and secreted IL‐6. The onset of IL‐6 production was rapid and prolonged. It was detected as early as 2 h after injury and persisted for the entire period of 21 days tested after the injury. Of the non‐neuronal cells that reside in intact and injured nerves, macrophages and fibroblasts were the major contributors to IL‐6 production. We also studied IL‐6 production in intact and injured nerves of mutant C57BL/6‐WLD/OLA/NHSD mice, which display very slow progression of Wallerian degeneration. Injured nerves of C57BL/6‐WLD/OLA/NHSD mice produced significantly lower amounts of IL‐6 than did rapidly degenerating nerves of C57/BL/6NHSD mice.

Galectin-3/MAC-2 in experimental allergic encephalomyelitis.

The removal of degenerating myelin by phagocytosis is central to pathogenesis and repair in traumatized and diseased nervous system. Galectin-3/MAC-2 is a differentiation and activation marker of murine and human monocytes/macrophages/microglia. Galectin-3/MAC-2, along with MAC-1 that mediates myelin phagocytosis, marks an in vivo activation state in macrophages, which are involved in myelin degeneration and phagocytosis in injured mouse peripheral nerves. In contrast, high levels of MAC-1 but extremely low levels of Galectin-3/MAC-2 are expressed in vivo in injured CNS where myelin degeneration and phagocytosis progress extremely slowly. The present study was aimed at testing whether an activation state marked by Galectin-3/MAC-2 is present in vivo in the CNS of EAE mice concomitant with autoimmune induced myelin degeneration and phagocytosis. EAE was inflicted by mouse spinal cord homogenate. Demyelination was assessed by light microscopy and Galectin-3/MAC-2, MAC-1, and F4/80 expression by immunocytochemistry. We presently document that Galectin-3/MAC-2 expression is up regulated, along with MAC-1 and F4/80, in spinal cords and optic nerves of EAE mice in areas of demyelination and myelin degeneration, in myelin phagocytosing microglia and macrophages. Copolymer 1 (Glatiramer acetate) suppresses EAE, demyelination, and Galectin-3/MAC-2 expression. EAE pathogenesis thus involves a state of activation in microglia and macrophages characterized by the expression Galectin-3/MAC-2 along with MAC-1. Furthermore, the in vivo responses to injury and autoimmune challenge in the CNS differ in the activation pattern of microglia and macrophages with regard to Galectin-3/MAC-2 expression and the corresponding occurrence of myelin degeneration and phagocytosis.

The cytokine network of wallerian degeneration: IL-10 and GM-CSF.

Wallerian degeneration (WD) is the inflammatory response of peripheral nerves to injury. Evidence is provided that granulocyte macrophage colony stimulating factor (GM‐CSF) contributes to the initiation and progression of WD by activating macrophages and Schwann, whereas IL‐10 down‐regulates WD by inhibiting GM‐CSF production. A significant role of activated macrophages and Schwann for future regeneration is myelin removal by phagocytosis and degradation. We studied the timing and magnitude of GM‐CSF and IL‐10 production, macrophage and Schwann activation, and myelin degradation in C57BL/6NHSD and C57BL/6‐WLD/OLA/NHSD mice that display normal rapid‐WD and abnormal slow‐WD, respectively. We observed the following events in rapid‐WD. The onset of GM‐CSF production is within 5 h after injury. Production is steadily augmented during the first 3 days, but is attenuated thereafter. The onset of production of the macrophage and Schwann activation marker Galectin‐3/MAC‐2 succeeds that of GM‐CSF. Galectin‐3/MAC‐2 production is up‐regulated during the first 6 days, but is down‐regulated thereafter. The onset of myelin degradation succeeds that of Galectin‐3/MAC‐2, and is almost complete within 1 week. IL‐10 production displays two phases. An immediate low followed by a high that begins on the fourth day, reaching highest levels on the seventh. The timing and magnitude of GM‐CSF production thus enable the rapid activation of macrophages and Schwann that consequently phagocytose and degrade myelin. The timing and magnitude of IL‐10 production suggest a role in down‐regulating WD after myelin is removed. In contrast, slow‐WD nerves produce low inefficient levels of GM‐CSF and IL‐10 throughout. Therefore, deficient IL‐10 levels cannot account for inefficient GM‐CSF production, whereas deficient GM‐CSF levels may account, in part, for slow‐WD.

Complement-receptor-3 and scavenger-receptor-AI/II mediated myelin phagocytosis in microglia and macrophages.

Microglia and macrophages express the alpha(M)/beta(2) integrin complement-receptor-3 (CR3/MAC-1; CD11b/CD18) and scavenger-receptor-AI/II (SRAI/II). Both can mediate myelin phagocytosis. We document that CR3/MAC-1 mediated myelin phagocytosis in microglia is modulated by complement and anti-CR3/MAC-1 mAbs. Complement augmented phagocytosis twofold. Anti-alpha(M) mAbs M1/70 and 5C6 inhibited and anti-beta(2) mAb M18/2 augmented myelin phagocytosis in the presence and absence of active complement. Active complement modulated phagocytosis inhibition by M1/70 and 5C6 and phagocytosis augmentation by M18/2. CR3/MAC-1 mediated myelin phagocytosis may thus be, at least partially, independent of but modulated by complement. Anti-beta(2) mAb Game-46 did not affect phagocytosis. However, combining M18/2 with Game-46 resulted in phagocytosis augmentation that was larger in magnitude than that induced by M18/2 alone. Thus, phagocytosis augmentation induced by one anti-beta(2) mAb was potentiated by another anti-beta(2) mAb. Combining M1/70 or 5C6 with M18/2 inhibited M18/2-induced augmentation. Overall, mAbs-induced phagocytosis modulation ranged three- to sevenfold from inhibition to augmentation. Anti-CR3/MAC-1 mAbs may reveal a mechanism by which native extracellular molecules bind to and modulate CR3/MAC-1 mediated myelin phagocytosis in microglia and macrophages. We further document SRAI/II mediated myelin phagocytosis in microglia and CR3/MAC-1 contributing to myelin phagocytosis two- to threefold more than SRAI/II when the two receptors function together.

Galectin‐3/MAC‐2, Ras and PI3K activate complement receptor‐3 and scavenger receptor‐AI/II mediated myelin phagocytosis in microglia

The removal of degenerated myelin is essential for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). Microglia can remove degenerated myelin through phosphatidylinositol‐3‐kinase (PI3K)‐dependent phagocytosis mediated by complement receptor‐3 (CR3/MAC‐1) and scavenger receptor‐AI/II (SRAI/II). Paradoxically, these receptors are expressed in microglia after injury but myelin is not phagocytosed. Additionally, Galectin‐3/MAC‐2 is expressed in microglia that phagocytose but not in microglia that do not phagocytose, suggesting that Galectin‐3/MAC‐2 is instrumental in activating phagocytosis. S‐trans, trans‐farnesylthiosalicylic (FTS), which inhibits Galectin‐3/MAC‐2 dependent activation of PI3K through Ras, inhibited phagocytosis. K‐Ras‐GTP levels and PI3K activity increased during normal phagocytosis and decreased during FTS‐inhibited phagocytosis. Galectin‐3/MAC‐2, which binds and stabilizes active Ras, coimmunoprecipitated with Ras and levels of the coimmunoprecipitate increased during normal phagocytosis. A role for Galectin‐3/MAC‐2 dependent activation of PI3K through Ras, mostly K‐Ras, is thus suggested. An explanation may thus be offered for deficient phagocytosis by microglia that express CR3/MAC‐1 and SRAI/II without Galectin‐3/MAC‐2 and efficient phagocytosis when CR3/MAC‐1 and SRAI/II are co‐expressed with Galectin‐3/MAC‐2.

cAMP cascade (PKA, Epac, adenylyl cyclase, Gi, and phosphodiesterases) regulates myelin phagocytosis mediated by complement receptor-3 and scavenger receptor-AI/II in microglia and macrophages.

The removal by phagocytosis of degenerated myelin is central for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). We tested for roles played by the cAMP cascade in the regulation of myelin phagocytosis mediated by complement receptor‐3 (CR3/MAC‐1) and scavenger receptor‐AI/II (SRAI/II) separately and combined in mouse microglia and macrophages. Components of the cAMP cascade tested are cAMP, adenylyl cyclase (AC), Gi, protein kinase A (PKA), exchange protein directly activated by cAMP (Epac), and phosphodiesterases (PDE). PKA inhibitors H‐89 and PKI14‐22 amide inhibited phagocytosis at normal operating cAMP levels (i.e., those occurring in the absence of reagents that alter cAMP levels), suggesting activation of phagocytosis through PKA at normal cAMP levels. Phagocytosis was inhibited by reagents that elevate endogenous cAMP levels to above normal: Gi‐inhibitor Pertussis toxin (PTX), AC activator Forskolin, and PDE inhibitors IBMX and Rolipram. Phagocytosis was inhibited also by cAMP analogues whose addition mimics abnormal elevations in endogenous cAMP levels: nonselective 8‐bromo‐cAMP, PKA‐specific 6‐Benz‐cAMP, and Epac‐specific 8‐CPT‐2′‐O‐Me‐cAMP, suggesting that abnormal high cAMP levels inhibit phagocytosis through PKA and Epac. Altogether, observations suggest a dual role for cAMP and PKA in phagocytosis: activation at normal cAMP levels and inhibition at higher. Furthermore, a balance between Gi‐controlled cAMP production by AC and cAMP degradation by PDE maintains normal operating cAMP levels that enable efficient phagocytosis. ©2005 Wiley‐Liss, Inc.

Distinct Inflammatory Stimuli Induce Different Patterns of Myelin Phagocytosis and Degradation in Recruited Macrophages

Injury and demyelinating diseases result in the disruption of the myelin sheath that surrounds axons in the nervous system. The removal of degenerating myelin by macrophages and microglia is central to repair mechanisms that follow. The efficiency of myelin removal depends on magnitudes and rates of myelin phagocytosis and degradation. In the present study we test whether environmental conditions within a tissue can control patterns of myelin removal. We document that macrophages that are recruited to the same tissue but by distinct inflammatory stimuli differ in their ability to phagocytose and degrade myelin. These observations may apply to the nervous system where different pathological conditions that involve distinct inflammatory stimuli may induce different functional states in microglia and macrophages.

Cytoskeleton plays a dual role of activation and inhibition in myelin and zymosan phagocytosis by microglia

A major innate immune function of microglia in the central nervous system is receptor‐mediated phagocytosis of tissue debris and pathogens. We studied how phagocytosis of degenerated myelin (i.e., tissue debris) and zymosan (i.e., yeast pathogen) is regulated by the cytoskeleton through myosin light chain kinase (MLCK) and the small GTPase Rho and its effector Rho‐kinase (ROCK) in primary mouse microglia. Our observations suggest a dual role of activation and inhibition of phagocytosis by MLCK and Rho/ ROCK signaling. MLCK activated, whereas Rho/ROCK down‐regulated complement receptor‐3 (CR3) mediated, phagocytosis of C3bi‐opsonized and nonopsonized myelin. These opposing roles of MLCK and Rho/ROCK depended on the preferential spatial localization of their distinctive functions. MLCK further activated, and Rho/ROCK down‐regulated, phagocytosis of nonopsonized zymosan by nonopsonic receptors (e.g., Dectin‐1). In contrast, MLCK down‐regulated, but Rho/ROCK activated, CR3‐mediated phagocytosis of C3bi‐opsonized zymosan. Thus MLCK and Rho/ROCK can each activate or inhibit phagocytosis but always act in opposition. Whether activation or inhibition occurs depends on the nature of the phagocytosed particle (C3bi‐opsonized or nonopsonized myelin or zymosan) and the receptors mediating each phagocytosis.—Gitik, M., Reichert, F., Rotshenker, S. Cytoskeleton plays a dual role of activation and inhibition in myelin and zymosan phagocytosis by microglia. FASEB J. 24, 2211–2221 (2010). www.fasebj.org

Non-PKC DAG/Phorbol-Ester receptor(s) inhibit complement receptor-3 and nPKC inhibit scavenger receptor-AI/II-mediated myelin phagocytosis but cPKC, PI3k, and PLCγ activate myelin phagocytosis by both

Complement‐receptor‐3 (CR3/MAC‐1), scavenger‐receptor‐AI/II (SRAI/II), and Fcγ‐receptor (FcγR) can mediate myelin phagocytosis in macrophages and microglia. Paradoxically, after injury to CNS axons these receptors are expressed but myelin is not phagocytosed, suggesting that phagocytosis is subject to regulation between efficient and inefficient states. In the present work, we focus on CR3/MAC‐1 and SRAI/II‐mediated myelin phagocytosis. Phagocytosis by CR3/MAC‐1 and SRAI/II was inhibited by cPKC inhibitor Go‐6976, general‐PKC inhibitors Ro‐318220 and calphostin‐C, and BAPTA/AM, which chelates intracellular Ca2+ required for cPKC activation. Signaling/activation by cPKC are thus suggested. PMA, which mimics diacylglycerol (DAG) as an activator of cPKC, novel‐PKC (nPKC), and non‐PKC DAG‐driven molecule(s), produced a dose‐dependent dual effect on phagocytosis by CR3/MAC‐1 and SRAI/II, i.e., augmentation at low concentrations and inhibition at high concentrations. Inhibition of phagocytosis by CR3/MAC‐1 was enhanced by combining inhibiting concentrations of PMA with PKC inhibitors Go‐6976 or Ro‐318220, suggesting inhibition by PMA/DAG‐driven non‐PKC molecule(s). In contrast, inhibition of phagocytosis by SRAI/II was enhanced by combining inhibiting concentrations of PMA with cPKC inhibitor Go‐6976 but not with general‐PKC inhibitor Ro‐318220, suggesting inhibition by nPKC. Phagocytosis by CR3/MAC‐1 and SRAI/II was further inhibited by PI3K inhibitors wortmannin and LY‐294002 and PLCγ inhibitor U‐73122. Altogether, our observations suggest that CR3/MAC‐1 and SRAI/II‐mediated myelin phagocytosis share activation by PI3K, PLCγ and cPKC. The two differ, however, in that non‐PKC DAG‐driven molecule(s) inhibit CR3/MAC‐1‐mediated phagocytosis, whereas nPKC inhibit SRAI/II‐mediated phagocytosis. Each of these signaling steps may be targeted for regulating CR3/MAC‐1 and/or SRAI/II‐mediated phagocytosis between efficient and inefficient states.