Adhesion molecules of the immunoglobulin superfamily are crucial effectors of leukocyte trafficking into the central nervous system. Using a lipid raft-based proteomic approach, we identified ALCAM as an adhesion molecule involved in leukocyte migration across the blood-brain barrier (BBB). ALCAM expressed on BBB endothelium localized together with CD6 on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was upregulated in active multiple sclerosis and experimental autoimmune encephalomyelitis lesions. Moreover, ALCAM blockade restricted the transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo and reduced the severity and delayed the time of onset of experimental autoimmune encephalomyelitis. Our findings indicate an important function for ALCAM in the recruitment of leukocytes into the brain and identify ALCAM as a potential target for the therapeutic dampening of neuroinflammation.

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model of multiple sclerosis (MS), an inflammatory, demyelinating disease of the central nervous system (CNS). EAE pathogenesis involves various cell types, cytokines, chemokines, and adhesion molecules. Given the complexity of the inflammatory response in EAE, it is likely that many immune mediators still remain to be discovered. To identify novel immune mediators of EAE pathogenesis, we performed an Affymetrix gene array screen on the spinal cords of mice at the onset stage of disease. This screening identified the gene encoding lipocalin 2 (Lcn2) as being significantly upregulated. Lcn2 is a multi-functional protein that plays a role in glial activation, matrix metalloproteinase (MMP) stabilization, and cellular iron flux. As many of these processes have been implicated in EAE, we characterized the expression and role of Lcn2 in this disease in C57BL/6 mice. We show that Lcn2 is significantly upregulated in the spinal cord throughout EAE and is expressed predominantly by monocytes and reactive astrocytes. The Lcn2 receptor, 24p3R, is also expressed on monocytes, macrophages/microglia, and astrocytes in EAE. In addition, we show that EAE severity is increased in Lcn2(-/-) mice as compared with wild-type controls. Finally, we demonstrate that elevated levels of Lcn2 are detected in the plasma and cerebrospinal fluid (CSF) in MS and in immune cells in CNS lesions in MS tissue sections. These data indicate that Lcn2 is a modulator of EAE pathogenesis and suggest that it may also play a role in MS.

Lipocalin 2 (Lcn2) plays an important role in defense against bacterial infection by interfering with bacterial iron acquisition. Although Lcn2 is expressed in a number of aseptic inflammatory conditions, its role in these conditions remains unclear. We examined the expression and role of Lcn2 after spinal cord injury (SCI) in adult mice by using a contusion injury model. Lcn2 expression at the protein level is rapidly increased 12-fold at 1 d after SCI and decreases gradually thereafter, being three times as high as control levels at 21 d after injury. Lcn2 expression is strongly induced after contusion injury in astrocytes, neurons, and neutrophils. The Lcn2 receptor (Lcn2R), which has been shown to influence cell survival, is also expressed after SCI in the same cell types. Lcn2-deficient (Lcn2 ؊/؊ ) mice showed significantly better locomotor recovery after spinal cord contusion injury than wild-type (Lcn2 ؉/؉ ) mice. Histological assessments indicate improved neuronal and tissue survival and greater sparing of myelin in Lcn2 ؊/؊ mice after contusion injury. Flow cytometry showed a decrease in neutrophil influx and a small increase in the monocyte population in Lcn2 ؊/؊ injured spinal cords. This change was accompanied by a reduction in the expression of several pro-inflammatory chemokines and cytokines as well as inducible nitric oxide synthase early after SCI in Lcn2 ؊/؊ mice compared with wild-type animals. Our results, therefore, suggest a role for Lcn2 in regulating inflammation in the injured spinal cord and that lack of Lcn2 reduces secondary damage and improves locomotor recovery after spinal cord contusion injury.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system (CNS). Like MS, the animal model experimental autoimmune encephalomyelitis (EAE) is characterized by CNS inflammation and demyelination and can follow a relapsing-remitting (RR) or chronic (CH) disease course. The molecular and pathological differences that underlie these different forms of EAE are not fully understood. We have compared the differences in RR- and CH-EAE generated in the same mouse strain (C57BL/6) using the same antigen. At the peak of disease when mice in both groups have similar clinical scores, CH-EAE is associated with increased lesion burden, myelin loss, axonal damage, and chemokine/cytokine expression when compared with RR-EAE. We further showed that inflammation and myelin loss continue to worsen in later stages of CH-EAE, whereas these features are largely resolved at the equivalent stage in RR-EAE. Additionally, axonal loss at these later stages is more severe in CH-EAE than in RR-EAE. We also demonstrated that CH-EAE is associated with a greater predominance of CD8(+) T cells in the CNS that exhibit MOG(35-55) antigen specificity. These studies therefore showed that, as early as the peak stage of disease, RR- and CH-EAE differ remarkably in their immune cell profile, chemokine/cytokine responses, and histopathological features. These data also indicated that this model of CH-EAE exhibits pathological features of a chronic-progressive disease profile and suggested that the sustained chronic phenotype is due to a combination of axonal loss, myelin loss, and continuing inflammation.

No abstract

The phagocytic cell response within the injured spinal cord is inefficient, allowing myelin debris to remain for prolonged periods of time within white matter tracts distal to the injury. Several proteins associated with this degenerating myelin are inhibitory to axon growth and therefore prevent severed axons from regenerating. Inflammatory agents such as lipopolysaccharide (LPS) can stimulate both the migration and phagocytic activity of macrophages. Using in situ hybridization, we found that the expression of the LPS membrane receptor, CD14, was enhanced in the mouse dorsal column following a dorsal hemisection. Double labeling studies showed that microglia and macrophages are the two major cell types expressing CD14 mRNA following spinal cord injury (SCI). We therefore tested whether systemic injections of LPS would increase the number and phagocytic activity of macrophages/microglia in the ascending sensory tract (AST) of the mouse dorsal column following a dorsal hemisection. Mice were treated daily via intraperitoneal injections of either LPS or phosphate-buffered saline (PBS). At 7 days post-SCI, greater numbers of activated mononuclear phagocytes were present in the AST undergoing Wallerian degeneration (WD) in LPS-treated animals compared with controls. Animals treated with LPS also exhibited greater Oil Red O staining, which is specific for degenerating myelin and macrophages phagocytosing myelin debris. Myelin clearance was confirmed at 7 days using Luxol Fast Blue staining and on toluidine blue-stained semi-thin sections. These results indicate that it is possible to manipulate the innate immune response to accelerate myelin clearance during WD in the injured mouse spinal cord.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated neuroinflammatory disease that is often used as a model of multiple sclerosis. EAE can follow either relapsing-remitting (RR) or chronic (CH) courses, yet the factors responsible for differentially inducing these forms of disease remain largely unknown. Proinflammatory cytokines play an important role in EAE, and signaling by these cytokines can be negatively regulated by the suppressor of cytokine signaling 1 protein (SOCS1). We assessed if differential expression of SOCS1 could contribute to the clinical course of RR and CH forms of EAE induced in the same mouse strain (C57BL/6) using the same myelin antigen (myelin oligodendrocyte glycoprotein). We show that SOCS1 mRNA levels are significantly elevated in the spinal cord in early stages of both RR- and CH-EAE. SOCS1 protein is highly expressed in immune cells in EAE lesions in the spinal cord, with expression predominantly localized within macrophages. Importantly, the number of Mac-1(+) macrophages expressing SOCS1 at the peak stage of RR disease is three-fold greater than in CH-EAE. Furthermore, the macrophage effector molecule iNOS, whose expression is regulated by SOCS1, is significantly reduced at the peak of RR- versus CH-EAE. Finally, the administration of a SOCS1-mimetic peptide reduces disease severity in the CH-EAE model. Thus, the differential expression of SOCS1 may contribute to the development of RR and CH forms of EAE.

Daclizumab and basiliximab, engineered human IgG monoclonal antibodies to the interleukin-2 (IL-2) receptor alpha-subunit, were approved to prevent acute rejection after renal transplantation. Daclizumab was studied in adult and pediatric renal allograft recipients, liver allograft recipients, and calcineurin-sparing protocols in renal transplant recipients. Basiliximab was studied in renal allograft recipients and subgroups of recipients of living-related and cadaveric transplants, and in patients with diabetes mellitus. Both agents reduced acute rejection and were associated with few adverse effects. However, information regarding their long-term effects on infection, malignancy, chronic rejection, and patient survival must be available before a final decision is made regarding their proper administration. We propose that a likely role the drugs will play in the field of solid organ transplantation is in new protocols that allow sparing of other more toxic immunosuppressive agents.