The mitochondrial Nod-like receptor NLRX1 modifies apoptosis through SARM1.

NLRX1, the mitochondrial NOD-like receptor (NLR), modulates apoptosis in response to both intrinsic and extrinsic cues. Insights into the mechanism of how NLRX1 influences apoptosis remain to be determined. Here, we demonstrate that NLRX1 associates with SARM1, a protein with a toll/interleukin-1 receptor (TIR)-containing domain also found in adaptor proteins downstream of toll-like receptors, such as MyD88. While a direct role of SARM1 in innate immunity is unclear, the protein plays essential roles in Wallerian degeneration (WD), a type of neuronal catabolism occurring following axonal severing or damage. In non-neuronal cells, we found that endogenous SARM1 was equally distributed in the cytosol and the mitochondrial matrix, where association with NLRX1 occurred. In these cells, the apoptotic role of NLRX1 was fully dependent on SARM1, indicating that SARM1 was downstream of NLRX1 in apoptosis regulation. In primary murine neurons, however, Wallerian degeneration induced by vinblastine or NGF deprivation occurred in SARM1- yet NLRX1-independent manner, suggesting that WD requires the cytosolic pool of SARM1 or that NLRX1 levels in neurons are too low to contribute to WD regulation. Together, these results shed new light into the mechanisms through which NLRX1 controls apoptosis and provides evidence of a new link between NLR and TIR-containing proteins.

Neuroimmune processes associated with Wallerian degeneration support neurotrophin-3-induced axonal sprouting in the injured spinal cord.

Lesions of the spinal cord cause two distinctive types of neuroimmune responses, a response at the lesion site that leads to additional tissue destruction and a more subtle response, termed Wallerian degeneration (WD), that occurs distal to the lesion site. We have evidence that the neuroimmune response associated with WD may support tissue repair. Previously, we found that overexpression of neurotrophin-3 (NT-3) induced axonal growth in the spinal cord after a unilateral corticospinal tract (CST) lesion, but only if the immune system was intact and activated. We reasoned that a neuroimmune response associated with WD was involved in this neuroplasticity. To test this, we compared NT-3-induced axonal sprouting in athymic nude rats that lack functional T cells with rats with functional T cells and in nude rats grafted with CD4(+) T cells or CD8(+) T cells. There was no sprouting in nude rats and in nude rats grafted with CD8(+) T cells. However, nude rats grafted with CD4(+) T cells mounted a sprouting response. To determine which CD4(+) subtype, type 1 T helper (Th1) or type 2 T helper (Th2) cells, was responsible, we grafted Th1 and Th2 cells into nude rats and tested whether they would support sprouting. Axonal sprouting was greater in rats grafted with Th2 cells, demonstrating that the Th2 subtype was responsible for supporting axonal sprouting. These data suggest that WD activates Th2 cells that, along with the direct effects of NT-3 on CST axons, act to support axonal sprouting in the lesioned spinal cord.

Acute injury in the peripheral nervous system triggers an alternative macrophage response.

BACKGROUND: The activation of the immune system in neurodegeneration has detrimental as well as beneficial effects. Which aspects of this immune response aggravate the neurodegenerative breakdown and which stimulate regeneration remains an open question. To unravel the neuroprotective aspects of the immune system we focused on a model of acute peripheral nerve injury, in which the immune system was shown to be protective. METHODS: To determine the type of immune response triggered after axotomy of the sciatic nerve, a model for Wallerian degeneration in the peripheral nervous system, we evaluated markers representing the two extremes of a type I and type II immune response (classical vs. alternative) using real-time quantitative polymerase chain reaction (RT-qPCR), western blot, and immunohistochemistry. RESULTS: Our results showed that acute peripheral nerve injury triggers an anti-inflammatory and immunosuppressive response, rather than a pro-inflammatory response. This was reflected by the complete absence of classical macrophage markers (iNOS, IFN γ, and IL12p40), and the strong up-regulation of tissue repair markers (arginase-1, Ym1, and Trem2). The signal favoring the alternative macrophage environment was induced immediately after nerve damage and appeared to be established within the nerve, well before the infiltration of macrophages. In addition, negative regulators of the innate immune response, as well as the anti-inflammatory cytokine IL-10 were induced. The strict regulation of the immune system dampens the potential tissue damaging effects of an over-activated response. CONCLUSIONS: We here demonstrate that acute peripheral nerve injury triggers an inherent protective environment by inducing the M2 phenotype of macrophages and the expression of arginase-1. We believe that the M2 phenotype, associated with a sterile inflammatory response and tissue repair, might explain their neuroprotective capacity. As such, shifting the neurodegeneration-induced immune responses towards an M2/Th2 response could be an important therapeutic strategy.

Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury.

In this review, we first provide a brief historical perspective, discussing how peripheral nerve injury (PNI) may have caused World War I. We then consider the initiation, progression, and resolution of the cellular inflammatory response after PNI, before comparing the PNI inflammatory response with that induced by spinal cord injury (SCI).In contrast with central nervous system (CNS) axons, those in the periphery have the remarkable ability to regenerate after injury. Nevertheless, peripheral nervous system (PNS) axon regrowth is hampered by nerve gaps created by injury. In addition, the growth-supportive milieu of PNS axons is not sustained over time, precluding long-distance regeneration. Therefore, studying PNI could be instructive for both improving PNS regeneration and recovery after CNS injury. In addition to requiring a robust regenerative response from the injured neuron itself, successful axon regeneration is dependent on the coordinated efforts of non-neuronal cells which release extracellular matrix molecules, cytokines, and growth factors that support axon regrowth. The inflammatory response is initiated by axonal disintegration in the distal nerve stump: this causes blood-nerve barrier permeabilization and activates nearby Schwann cells and resident macrophages via receptors sensitive to tissue damage. Denervated Schwann cells respond to injury by shedding myelin, proliferating, phagocytosing debris, and releasing cytokines that recruit blood-borne monocytes/macrophages. Macrophages take over the bulk of phagocytosis within days of PNI, before exiting the nerve by the circulation once remyelination has occurred. The efficacy of the PNS inflammatory response (although transient) stands in stark contrast with that of the CNS, where the response of nearby cells is associated with inhibitory scar formation, quiescence, and degeneration/apoptosis. Rather than efficiently removing debris before resolving the inflammatory response as in other tissues, macrophages infiltrating the CNS exacerbate cell death and damage by releasing toxic pro-inflammatory mediators over an extended period of time. Future research will help determine how to manipulate PNS and CNS inflammatory responses in order to improve tissue repair and functional recovery.

Wallerian degeneration: the innate-immune response to traumatic nerve injury.

Traumatic injury to peripheral nerves results in the loss of neural functions. Recovery by regeneration depends on the cellular and molecular events of Wallerian degeneration that injury induces distal to the lesion site, the domain through which severed axons regenerate back to their target tissues. Innate-immunity is central to Wallerian degeneration since innate-immune cells, functions and molecules that are produced by immune and non-immune cells are involved. The innate-immune response helps to turn the peripheral nerve tissue into an environment that supports regeneration by removing inhibitory myelin and by upregulating neurotrophic properties. The characteristics of an efficient innate-immune response are rapid onset and conclusion, and the orchestrated interplay between Schwann cells, fibroblasts, macrophages, endothelial cells, and molecules they produce. Wallerian degeneration serves as a prelude for successful repair when these requirements are met. In contrast, functional recovery is poor when injury fails to produce the efficient innate-immune response of Wallerian degeneration.

Initiation and progression of axonopathy in experimental autoimmune encephalomyelitis.

Axonal loss is the principal cause of chronic disability in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). In C57BL/6 mice with EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide 35-55, the first evidences of axonal damage in spinal cord were in acute subpial and perivascular foci of infiltrating neutrophils and lymphocytes and included intra-axonal accumulations of the endovesicular Toll-like receptor TLR8, and the inflammasome protein NAcht leucine-rich repeat protein 1 (NALP1). Later in the course of this illness, focal inflammatory infiltrates disappeared from the spinal cord, but there was persistent activation of spinal cord innate immunity and progressive, bilaterally symmetric loss of small-diameter corticospinal tract axons. These results support the hypothesis that both contact-dependent and paracrine interactions of systemic inflammatory cells with axons and an innate immune-mediated neurodegenerative process contribute to axonal loss in this multiple sclerosis model.

Toll-like receptor expression in the peripheral nerve.

Toll-like receptors comprise a family of evolutionary conserved pattern recognition receptors that act as a first defense line in the innate immune system. Upon stimulation with microbial ligands, they orchestrate the induction of a host defense response by activating different signaling cascades. Interestingly, they appear to detect the presence of endogenous signals of danger as well and as such, neurodegeneration is thought to trigger an immune response through ligation of TLRs. Though recent data report the expression of various TLRs in the central nervous system, TLR expression patterns in the peripheral nervous system have not been determined yet. We observed that Schwann cells express relatively high levels of TLRs, with especially TLR3 and TLR4 being prominent. Sensory and motor neurons hardly express TLRs at all. Through the use of NF-κB signaling as read-out, we could show that all TLRs are functional in Schwann cells and that bacterial lipoprotein, a ligand for TLR1/TLR2 receptors yields the strongest response. In sciatic nerve, basal levels of TLRs closely reflect the expression patterns as determined in Schwann cells. TLR3, TLR4, and TLR7 are majorly expressed, pointing to their possible role in immune surveillance. Upon axotomy, TLR1 becomes strongly induced, while most other TLR expression levels remain unaffected. Altogether, our data suggest that similar to microglia in the brain, Schwann cells might act as sentinel cells in the PNS. Furthermore, acute neurodegeneration induces a shift in TLR expression pattern, most likely illustrating specialized functions of TLRs in basal versus activated conditions of the peripheral nerve.

Toll-like receptors in peripheral nerve injury and neuropathic pain.

Peripheral nerve injury triggers a series of responses in the injured nerve, such as the dissolution of distal axons, the activation of Schwann cells, the production of various proinflammatory mediators, and the infiltration of circulating immune cells. These orchestrated events regulate the degeneration and subsequent regeneration of the injured nerve. In addition, peripheral nerve injury often accompanies chronic pain. Studies in this field have revealed that spinal cord microglia activation plays a critical role in the development of pain hypersensitivity. Recent studies using genetically modified mice indicate that Toll-like receptors (TLRs) are involved in nerve degeneration (Wallerian degeneration) and chronic pain (neuropathic pain) development after nerve injury. Here, we review studies that have implicated TLRs in mediating nerve degeneration/regeneration and neuropathic pain following nerve injury. In addition, we discuss possible mechanisms underlying the roles of TLRs in these neurological disorders.

Diabetic neuropathy: electrophysiological and morphological study of peripheral nerve degeneration and regeneration in transgenic mice that express IFNbeta in beta cells.

Diabetic neuropathy is one of the most frequent complications in diabetes but there are no treatments beyond glucose control, due in part to the lack of an appropriate animal model to assess an effective therapy. This study was undertaken to characterize the degenerative and regenerative responses of peripheral nerves after induced sciatic nerve damage in transgenic rat insulin I promoter / human interferon beta (RIP/IFNbeta) mice made diabetic with a low dose of streptozotocin (STZ) as an animal model of diabetic complications. In vivo, histological and immunohistological studies of cutaneous and sciatic nerves were performed after left sciatic crush. Functional tests, cutaneous innervation, and sciatic nerve evaluation showed pronounced neurological reduction in all groups 2 weeks after crush. All animals showed a gradual recovery but this was markedly slower in diabetic animals in comparison with normoglycemic animals. The delay in regeneration in diabetic RIP/IFNbeta mice resulted in an increase in active Schwann cells and regenerating neurites 8 weeks after surgery. These findings indicate that diabetic-RIP/IFNbeta animals mimic human diabetic neuropathy. Moreover, when these animals are submitted to nerve crush they have substantial deficits in nerve regrowth, similar to that observed in diabetic patients. When wildtype animals were treated with the same dose of STZ, no differences were observed with respect to nontreated animals, indicating that low doses of STZ and the transgene are not implicated in development of the degenerative and regenerative events observed in our study. All these findings indicate that RIP/IFNbeta transgenic mice are a good model for diabetic neuropathy.

Molecular inflammatory mediators in peripheral nerve degeneration and regeneration.

Wallerian degeneration, the self-destructive set of cellular and molecular processes by which degenerating axons and myelin are cleared after injury, is initiated by macrophages and Schwann cells. Molecular inflammatory mediators such as cytokines (IL-1, IL-6, IL-10, and TNF-alpha, among others), transcription factors (NF-kappaB, c-Jun), the complement system and arachidonic acid metabolites have been shown to modulate these processes in various studies. However, the exact role that each of these mediators plays during axonal degeneration and regeneration has not been fully established. Understanding the molecular basis of these interactions between the immune system and peripheral nerve injury would open the possibility of targeting these inflammatory mediators as therapeutic interventions. In this review we attempt to integrate the current evidence generated around this issue, and to explore the therapeutic possibilities that arise.

Neuroprotection without immunomodulation is not sufficient to reduce first relapse severity in experimental autoimmune encephalomyelitis.

OBJECTIVES: Multiple sclerosis can be characterized by a strong neuroinflammatory and progressive neurodegenerative component leading to prolonged disability. The synthetic compound R(+)WIN55,212-2 is reported to be neuroprotective at moderate doses and both neuroprotective and immunomodulatory at high doses, most likely due to differences in receptor affinities. In order to investigate the effects of neuroprotection and immunomodulation in an animal model of multiple sclerosis, we examined the impact of increasing concentrations of R(+)WIN55,212-2 on the inflammatory profile in CNS during first relapse and related this to demyelination, axonal degeneration and relapse severity. METHODS: Experimental autoimmune encephalomyelitis was induced in Dark Agouti rats and treatment with R(+)WIN55,212-2 was initiated at symptom debut. The animals were scored clinically throughout the experiment, and axonal degeneration, demyelination, T cells, microglia/macrophages, TNF-alpha, IL-12, IFN-gamma, IL-10 and the T(H)17 response were estimated at the peak of the first relapse. RESULTS: Treatment with high-dose R(+)WIN55,212-2 (10 and 20 mg/kg) significantly improved the clinical performance of the animals during relapse. Interestingly, treatment at any dosage did not affect the brain levels of TNF-alpha, IL-12 and IFN-gamma (T(H)1 response), whereas high-dose cannabinoid treatment reduced the number of T cells and microglia/macrophages in addition to the T(H)17 response. At the same time, we observed a significant reduction in axonal degeneration in all treatment groups whereas only high-dose treatment resulted in reduced demyelination. CONCLUSION: High-dose R(+)WIN55,212-2 treatment reduces demyelination and axonal degeneration and has immunomodulatory effects which significantly improve clinical performance, whereas a reduction in axonal degeneration on its own, induced by 5 mg/kg R(+)WIN55,212-2, has no impact on first relapse severity.

Imaging resident and recruited macrophage contribution to Wallerian degeneration.

Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MPs) of the peripheral nervous system (PNS) are the main cellular agent controlling this process. Some evidence suggests that resident PNS-MPs along with MPs of hematogenous origin may be involved, but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporter mice and coherent anti-Stokes Raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatiotemporal choreography of resident and recently recruited MPs after injury and unveiled distinct functions of these subsets, with recruited MPs being responsible for efficient myelin stripping and clearance and resident MPs being involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases.

Detection of Neutrophils in the Sciatic Nerve Following Peripheral Nerve Injury.

Injury to the sciatic nerve leads to degeneration and debris clearance in the area distal to the injury site, a process known as Wallerian degeneration. Immune cell infiltration into the distal sciatic nerve plays a major role in the degenerative process and subsequent regeneration of the injured motor and sensory axons. While macrophages have been implicated as the major phagocytic immune cell participating in Wallerian degeneration, recent work has found that neutrophils, a class of short-lived, fast responding white blood cells, also significantly contribute to the clearance of axonal and myelin debris. Detection of specific myeloid subtypes can be difficult as many cell-surface markers are often expressed on both neutrophils and monocytes/macrophages. Here we describe two methods for detecting neutrophils in the axotomized sciatic nerve of mice using immunohistochemistry and flow cytometry. For immunohistochemistry on fixed frozen tissue sections, myeloperoxidase and DAPI are used to specifically label neutrophils while a combination of Ly6G and CD11b are used to assess the neutrophil population of unfixed sciatic nerves using flow cytometry.

In Vivo Analysis of Glial Immune Responses to Axon Degeneration in Drosophila melanogaster.

Axon degeneration elicits a range of immune responses from local glial cells, including striking changes in glial gene expression, morphology, and phagocytic activity. Here, we describe a detailed set of protocols to assess discrete components of the glial reaction to axotomy in the adult nervous system of Drosophila melanogaster. These methods allow one to visualize and quantify transcriptional, morphological, and functional responses of glia to degenerating axons in a model system that is highly amenable to genetic manipulation.

Immunoglobulin M deposition in cutaneous nerves of anti-myelin-associated glycoprotein polyneuropathy patients correlates with axonal degeneration.

Anti-myelin-associated glycoprotein (MAG) neuropathy is an antibody-mediated polyneuropathy. We correlated clinical features, immunoglobulin (Ig) M blood levels, IgM deposition and axonal degeneration in skin biopsies of anti-MAG neuropathy patients. By confocal microscopy, IgM deposits were found exclusively within perineurium-enclosed nerves; they were not found on single, non-perineurium-ensheathed myelinated axons. There was a linear correlation between IgM accumulation in nerve fascicles with IgM blood levels but not with anti-MAG antibody titer or disease duration. Axons with specific IgM deposits had signs of axonal damage, including neurofilament disintegration. Nodal structures were intact even at sites where the axons showed pathologic changes. Ultrastructural analysis revealed degeneration of myelinating Schwann cells. Taken together, these findings suggest that in anti-MAG neuropathy patients, IgM deposits are entrapped within cutaneous perineurium-ensheathed nerve bundles where they accumulate in the endoneurial space. High local IgM levels in the endoneurium may be required for IgM deposition on myelin and subsequent axonal injury and degeneration. This study underlines the importance of early, effective anti-B-cell treatments for preventing progression of this neuropathy.

Childhood steroid-responsive acute erythromelalgia with axonal neuropathy of large myelinated fibers: a dysimmune neuropathy?

A 12-year-old girl developed acute erythromelalgia of distal extremities. Physical, imaging and laboratory examinations failed to find an infective, systemic autoimmune, metabolic, endocrine, and vascular origin. The severe pain and allodynia indicated small-fiber neuropathy but muscle weakness suggested an involvement of large myelinated nerve fibers. This was confirmed by electrophysiological testing. High-dose then slowly tapered methylprednisolone resulted in rapid remission of painful erythromelalgia and complete electrophysiological recovery. Our case may suggest an additional variant to recently described steroid-responsive erythromelalgia with small-fiber axonopathy and may denote a transitory variant to Guillain-Barré syndrome or chronic dysimmune neuropathies.

The role of macrophages in optic nerve regeneration.

Following injury to the nervous system, the activation of macrophages, microglia, and T-cells profoundly affects the ability of neurons to survive and to regenerate damaged axons. The primary visual pathway provides a well-defined model system for investigating the interactions between the immune system and the nervous system after neural injury. Following damage to the optic nerve in mice and rats, retinal ganglion cells, the projection neurons of the eye, normally fail to regenerate their axons and soon begin to die. Induction of an inflammatory response in the vitreous strongly enhances the survival of retinal ganglion cells and enables these cells to regenerate lengthy axons beyond the injury site. T cells modulate this response, whereas microglia are thought to contribute to the loss of retinal ganglion cells in this model and in certain ocular diseases. This review discusses the complex and sometimes paradoxical actions of blood-borne macrophages, resident microglia, and T-cells in determining the outcome of injury in the primary visual pathway.

Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells.

Peripheral nerve injury is followed by Wallerian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose-specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes noneuronal cells, primarily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM-CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The proposed mechanism is based on the following novel observations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in intact nerves. The GM-CSF molecule was detected by ELISA in medium conditioned by in vitro and in vivo degenerating peripheral nerves as of the 4th h after injury. GM-CSF activity was demonstrated by two independent bioassays, and repressed by activity blocking antibodies. Significant levels of GM-CSF were produced by nerve derived fibroblasts, but neither by Schwann cells nor by nerve derived macrophages. Mouse rGM-CSF enhanced MAC-2 production in nerve explants, and up-regulated cell surface expression of MAC-2 by Schwann cells and macrophages. Interleukin-1 beta up-regulated GM-CSF production thus suggesting that injury induced GM-CSF production may be mediated by interleukin-1 beta. Our findings highlight the fact that fibroblasts, by producing GM-CSF and thereby affecting macrophage and Schwann function, play a significant role in the cascade of molecular events and cellular interactions of Wallerian degeneration.

Interactions between Schwann cells and macrophages in injury and inherited demyelinating disease.

In this article we first discuss the factors that regulate macrophage recruitment, activation, and myelin phagocytosis during Wallerian degeneration and some of the factors involved in the termination of inflammation at the end of the period of Wallerian degeneration after peripheral nerve injuries. In particular, we deal with the early events that trigger chemokine and cytokine expression; the role of phospholipase A(2) in initiating the breakdown of compact myelin, and chemokine, cytokine expression; and the role of MCP-1, MIP-1alpha, and IL-1beta in macrophage recruitment and myelin phagocytosis. We also discuss how inflammation may be switched off and the recently identified role of the Nogo receptor on activated macrophages in the clearance of these cells from the injured nerve. In the second half of the article we focus on the role of certain Schwann cell borne cytokines and chemokines, such as M-CSF and MCP-1 as well as intracellular signaling that regulate their expression in animal models of inherited demyelinating disease. Additionally, we present the preservation of sensory nerves fibers from macrophage attack in these animal models as a challenging paradigm for the development of putative treatment approaches. Finally, we also discuss the similarities and differences in these Schwann cell-macrophage responses in injury-induced Wallerian degeneration and inherited demyelinating diseases. Knowledge of the molecular mechanisms underlying Schwann cell-macrophage interaction under pathological conditions is an important prerequisite to develop effective treatment strategies for various peripheral nerve disorders.

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.