The encephalitogenic, human myelin oligodendrocyte glycoprotein-induced antibody repertoire is directed toward multiple epitopes in C57BL/6-immunized mice.

Although Abs specific for myelin oligodendrocyte glycoprotein (MOG) have been detected in patients with multiple sclerosis (MS), their contribution to pathogenesis remains poorly understood. Immunization of C57BL/6 mice with recombinant human MOG (hMOG) results in experimental autoimmune encephalomyelitis involving MOG-specific, demyelinating Abs. This model is therefore informative for understanding anti-MOG humoral responses in MS. In the current study, we have characterized the hMOG-specific Ab repertoire in immunized C57BL/6 mice using both in vitro and in vivo approaches. We demonstrate that hMOG-specific mAbs are not focused on one specific region of MOG, but instead target multiple epitopes. Encephalitogenicity of the mAbs, assessed by the ability of the mAbs to exacerbate experimental autoimmune encephalomyelitis in mice, correlates with the activity of the mAbs in binding to CNS tissue sections, but not with other in vitro assays. The targeting of different MOG epitopes by encephalitogenic Abs has implications for disease pathogenesis, because it could result in MOG cross linking on oligodendrocytes and/or immune complex formation. These studies reveal several novel features concerning pathogenic, humoral responses that may have relevance to human MS.

IFN-ß expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling.

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-ß. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-ß in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-ß mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-ß transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-ß promoter, revealed that IFN-ß mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-ß mRNA expression. Taken together, these data raise questions about the role of PKCε in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA.

Infection of neutrophil granulocytes with Leishmania major activates ERK 1/2 and modulates multiple apoptotic pathways to inhibit apoptosis.

Neutrophil granulocytes provide the first line of defense against bacterial, fungal, and parasitic infections. They phagocytose and kill many invading pathogens. Certain pathogenic microorganisms such as the intracellular protozoan parasite Leishmania major (L. major) can survive inside neutrophils. Mature neutrophils have a very short life span due to spontaneous apoptosis. Previously, we have reported that infections with L. major are able to delay spontaneous apoptosis. In the present study, we addressed the underlying mechanisms of regulation of both extrinsic and intrinsic apoptosis. We show that interaction with L. major transiently activates ERK1/2 phosphorylation. Pharmacological inhibition of ERK1/2 phosphorylation reversed the apoptosis delay. Moreover, infection leads to the enhanced and sustainable expression of the anti-apoptotic proteins Bcl-2 and Bfl-1, respectively. As downstream events, the release of cytochrome c from mitochondria and processing of caspase-6 were inhibited. We also confirm that infection with L. major results in reduced FAS expression on the surface of neutrophils. The presented data indicate that infection with L. major affects both intrinsic as well as extrinsic pathways of neutrophil apoptosis. Enhanced life span of host neutrophils enables the parasite to survive within neutrophils.

Phagocytosis of apoptotic cells by neutrophil granulocytes: diminished proinflammatory neutrophil functions in the presence of apoptotic cells.

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN-gamma results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF-alpha and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-alpha, which was independent of NF-kappaB and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.

Impairment of gamma interferon signaling in human neutrophils infected with Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the causative agent of tick-borne human granulocytic anaplasmosis (HGA), is an intracellular bacterium which survives and multiplies inside polymorphonuclear neutrophil granulocytes (PMN). Increased bacterial burden in gamma interferon (IFN-gamma)-deficient mice suggested a major role of IFN-gamma in the control of A. phagocytophilum. Here we investigated whether infection of human PMN with A. phagocytophilum impairs IFN-gamma signaling thus facilitating intracellular survival of the bacterium. The secretion of the IFN-gamma-inducible chemokines IP-10/CXCL10 and MIG/CXCL9 was markedly inhibited in infected neutrophils. Molecular analyses revealed that, compared to uninfected PMN, A. phagocytophilum decreased the expression of the IFN-gamma receptor alpha-chain CD119, diminished the IFN-gamma-induced phosphorylation of STAT1, and enhanced the expression of SOCS1 and SOCS3 in PMN. Since IFN-gamma activates various antibacterial effector mechanisms of PMN, the impaired IFN-gamma signaling in infected cells likely contributes to the survival of A. phagocytophilum inside PMN and to HGA disease development.

Autoantibody depletion ameliorates disease in murine experimental autoimmune encephalomyelitis.

Much data support a role for central nervous system antigen-specific antibodies in the pathogenesis of multiple sclerosis (MS). The effects of inducing a decrease in (auto)antibody levels on MS or experimental autoimmune encephalomyelitis (EAE) through specific blockade of FcRn, however, remain unexplored. We recently developed engineered antibodies that lower endogenous IgG levels by competing for binding to FcRn. These Abdegs ("antibodies that enhance IgG degradation") can be used to directly assess the effect of decreased antibody levels in inflammatory diseases. In the current study, we show that Abdeg delivery ameliorates disease in an EAE model that is antibody dependent. Abdegs could therefore have promise as therapeutic agents for MS.