Ruth Huizinga

Reactive oxygen species enhance the migration of monocytes across the blood-brain barrier in vitro.

Reactive oxygen species (ROS) are involved in the pathogenesis of several inflammatory and degenerative diseases, including multiple sclerosis (MS), an inflammatory disease of the central nervous system. We investigated the potential involvement of ROS in the interaction of monocytes with cerebral endothelium, because this is likely to be an early event in the development of MS lesions. ROS are produced via two main pathways, one involving NADPH oxidase complex and the other involving xanthine oxidase (XO). We examined the effects of ROS, ROS scavengers, and ROS inhibitors of both pathways on the migration of monocytes across the blood‐brain barrier in vitro. Scavengers and inhibitors of XO predominantly inhibited monocyte migration, whereas inhibitors and scavengers of the NADPH oxidase complex had no effect. Exposure of cerebral endothelial cells (CEC) to superoxide (O2−) resulted in enhanced migration and adhesion of monocytes as well as disruption of the tight junctions, whereas hydroxyl radicals and hydrogen peroxide induced no significant effect on these parameters. Underlying mechanisms of the observed changes were found to reside in the phospholipase C‐mediated signal transduction cascade, subsequent accumulation of inositol 1,4,5‐trisphosphate, and mobilization of intracellular calcium. We conclude that O2− is a signaling molecule that is produced during the firm adhesion of monocytes to CEC, which triggers cytoskeletal rearrangements allowing infiltration of monocytes into the brain.

Brain antigens in functionally distinct antigen-presenting cell populations in cervical lymph nodes in MS and EAE

Drainage of central nervous system (CNS) antigens to the brain-draining cervical lymph nodes (CLN) is likely crucial in the initiation and control of autoimmune responses during multiple sclerosis (MS). We demonstrate neuronal antigens within CLN of MS patients. In monkeys and mice with experimental autoimmune encephalomyelitis (EAE) and in mouse models with non-inflammatory CNS damage, the type and extent of CNS damage was associated with the frequencies of CNS antigens within the cervical lymph nodes. In addition, CNS antigens drained to the spinal-cord-draining lumbar lymph nodes. In human MS CLN, neuronal antigens were present in pro-inflammatory antigen-presenting cells (APC), whereas the majority of myelin-containing cells were anti-inflammatory. This may reflect a different origin of the cells or different drainage mechanisms. Indeed, neuronal antigen-containing cells in human CLN did not express the lymph node homing receptor CCR7, whereas myelin antigen-containing cells in situ and in vitro did. Nevertheless, CLN from EAE-affected CCR7-deficient mice contained equal amounts of myelin and neuronal antigens as wild-type mice. We conclude that the type and frequencies of CNS antigens within the CLN are determined by the type and extent of CNS damage. Furthermore, the presence of myelin and neuronal antigens in functionally distinct APC populations within MS CLN suggests that differential immune responses can be evoked.

Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis.

Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood‐borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well‐characterized antibodies to axonal antigens, that axonal damage is associated with HLA‐DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA‐DR+ cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF‐L), present in white and gray matter, was observed in human microglia. The number of NF‐L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF‐L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF‐L is taken up and degraded by the cell. In vivo, NF‐L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA‐DR+ cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens.

Surgical excision of CNS-draining lymph nodes reduces relapse severity in chronic-relapsing experimental autoimmune encephalomyelitis

Despite lack of classical lymphatic vessels in the central nervous system (CNS), cells and antigens do reach the CNS‐draining lymph nodes. These lymph nodes are specialized to mediate mucosal immune tolerance, but can also generate T‐ and B‐cell immunity. Their role in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) therefore remains elusive. We hypothesized that drainage of CNS antigens to the CNS‐draining lymph nodes is vital for the recurrent episodes of CNS inflammation. To test this, we surgically removed the superficial cervical lymph nodes, deep cervical lymph nodes, and the lumbar lymph nodes prior to disease induction in three mouse EAE models, representing acute, chronic, and chronic‐relapsing EAE. Excision of the CNS‐draining lymph nodes in chronic‐relapsing EAE reduced and delayed the relapse burden and EAE pathology within the spinal cord, which suggests initiation of CNS antigen‐specific immune responses within the CNS‐draining lymph nodes. Indeed, superficial cervical lymph nodes from EAE‐affected mice demonstrated proliferation against the immunizing peptide, and the deep cervical lymph nodes, lumbar lymph nodes, and spleen demonstrated additional proliferation against other myelin antigen epitopes. This indicates that intermolecular epitope spreading occurs and that CNS antigen‐specific immune responses are differentially generated within the different CNS‐draining lymphoid organs. Proliferation of splenocytes from lymphadenectomized and sham‐operated mice against the immunizing peptide was similar. These data suggest a role for CNS‐draining lymph nodes in the induction of detrimental immune responses in EAE relapses, and conclusively demonstrate that the tolerance‐inducing capability of cervical lymph nodes is not involved in EAE. Copyright

TLR4-Mediated Sensing of Campylobacter jejuni by Dendritic Cells Is Determined by Sialylation

In Guillain-Barré syndrome (GBS), ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) drives the production of cross-reactive Abs to peripheral nerve gangliosides. We determined whether sialic acid residues in C. jejuni LOS modulate dendritic cell (DC) activation and subsequent B cell proliferation as a possible mechanism for the aberrant humoral immune response in GBS. Highly purified sialylated LOS of C. jejuni isolates from three GBS patients induced human DC maturation and secretion of inflammatory cytokines that were inhibited by anti-TLR4 neutralizing Abs. The extent of TLR4 signaling and DC activation was greater with LOS of the wild type isolates than with nonsialylated LOS of the corresponding sialyltransferase gene knockout (cst-II mutant) strains, indicating that sialylation boosts the DC response to C. jejuni LOS. Supernatants of LOS-activated DCs induced B cell proliferation after cross-linking of surface Igs in the absence of T cells. Lower B cell proliferation indices were found with DC supernatants after DC stimulation with cst-II mutant or neuraminidase desialylated LOS. This study showed that sialylation of C. jejuni LOS enhances human DC activation and subsequent B cell proliferation, which may contribute to the development of cross-reactive anti-ganglioside Abs found in GBS patients following C. jejuni infection.

Human Adipose Tissue-Derived Mesenchymal Stem Cells Abrogate Plasmablast Formation and Induce Regulatory B Cells Independently of T Helper Cells

Mesenchymal or stromal stem cells (MSC) interact with cells of the immune system in multiple ways. Modulation of the immune system by MSC is believed to be a therapeutic option for autoimmune disease and transplant rejection. In recent years, B cells have moved into the focus of the attention as targets for the treatment of immune disorders. Current B‐cell targeting treatment is based on the indiscriminate depletion of B cells. The aim of this study was to examine whether human adipose tissue‐derived MSC (ASC) interact with B cells to affect their proliferation, differentiation, and immune function. ASC supported the survival of quiescent B cells predominantly via contact‐dependent mechanisms. Coculture of B cells with activated T helper cells led to proliferation and differentiation of B cells into CD19+CD27highCD38high antibody‐producing plasmablasts. ASC inhibited the proliferation of B cells and this effect was dependent on the presence of T cells. In contrast, ASC directly targeted B‐cell differentiation, independently of T cells. In the presence of ASC, plasmablast formation was reduced and IL‐10‐producing CD19+CD24highCD38high B cells, known as regulatory B cells, were induced. These results demonstrate that ASC affect B cell biology in vitro, suggesting that they can be a tool for the modulation of the B‐cell response in immune disease. Stem Cells 2015;33:880–891

Axonal loss and gray matter pathology as a direct result of autoimmunity to neurofilaments

Axonal damage is considered the major cause of irreversible disability in multiple sclerosis (MS). Which mechanisms underlie the damage and whether this is secondary to myelin damage remains to be clarified. Recently, we have demonstrated that autoimmunity to the axonal/neuronal cytoskeletal protein neurofilament light (NF-L) induces axonal damage and neurological disease including spasticity - a common feature of MS. To examine the relationship between axonal damage and demyelination we have characterized the detailed neuropathology of NF-L-induced disease in Biozzi mice compared to classical experimental autoimmune encephalomyelitis (EAE) induced with myelin oligodendrocyte glycoprotein (MOG). In NF-L-induced neurological disease the lesions were predominantly located in the dorsal column displaying extensive axonal degeneration, but were also abundant in the gray matter. In contrast, lesions in MOG-EAE were restricted to the lateral and ventral columns and displayed less axonal damage and little gray matter involvement. The differential lesion location was confirmed by quantitation of leukocyte subsets. In both diseases myelin damage was a common feature although the numerous empty myelin sheaths in NF-L-disease indicative of axonal damage suggest that myelin damage was a secondary event. In summary, autoimmunity to NF-L induces a distinct lesion topology, axonal damage and gray matter lesions supporting the notion that axonal loss and gray matter pathology can be the direct consequence of a primary autoimmune attack against axonal antigens such as NF-L rather than merely a secondary event to myelin damage.

Osteoprotegerin Is Associated With Aneurysm Diameter and Proteolysis in Abdominal Aortic Aneurysm Disease

Objective—Serum osteoprotegerin (OPG) concentrations have previously been associated with growth of abdominal aortic aneurysms (AAAs). In vitro experiments showed that OPG promotes matrix metalloprotease (MMP) release from monocytes and vascular smooth muscle cells. We hypothesized that OPG expression is increased in human AAAs and is associated with proteolysis. Methods and Results—AAA biopsies were collected from 329 patients. We assessed the concentrations of OPG, cathepsins A, B, and S as well as the activity of MMP-2 and MMP-9. The AAA wall infiltration by macrophages, lymphocytes, and plasma cells was estimated by immunohistochemistry. The concentration of OPG correlated positively with aortic diameter (<55 mm: 16.1 [5.8–28.7], 55–70 mm: 21.9 [10.2–36.0], >70 mm: 24.0 [13.5–52.9] ng OPG/mg total amount of protein, P=0.020), cathepsin A (r=0.221, P=0.005), B (r=0.384, P<0.001), and S (r=0.467, P<0.001), MMP-2 (r=0.180, P<0.001), MMP-9 (r=0.178, P<0.001), and the number of lymphocytes (P<0.001) and plasma cells (P=0.001). OPG immunostaining was predominantly demonstrated in plasma cells. Conclusion—The concentration of aortic wall OPG is positively associated with established markers of AAA severity and pathogenesis. OPG appeared to be associated with lymphocytes and plasma cells. These human data support previous experimental data suggesting a role for OPG in AAA pathogenesis.

IgG Fc N-Glycosylation in Guillain–Barré Syndrome Treated with Immunoglobulins

Intravenous immunoglobulin (IVIg) is the treatment of choice for Guillain-Barré syndrome (GBS), an immune-mediated peripheral neuropathy causing rapidly progressive limb weakness and respiratory failure. The working mechanism of IVIg in autoimmune diseases has not been elucidated, but previous studies indicate that some anti-inflammatory effects may be mediated by the N-glycosylation of the Fc-portion of IgG. GBS is a model disease to investigate these effects because GBS is an acute and monophasic disorder usually affecting healthy persons, which is treated with a standard course of IVIg, although the clinical response is highly variable. In the current study, the N-glycosylation of the Fc-portion of serum IgG was investigated in patients with GBS before and after treatment with IVIg in relation to clinical course and outcome. Glycoforms of serum IgG1 and IgG2 were determined separately by liquid chromatography mass spectrometry. These IgG subclasses were purified from the serum of 174 GBS patients before and in 150 patients 2 weeks after standard IVIg treatment regimen. Treatment-naive GBS patients compared with age- and sex-matched controls had lower levels of galactosylation of IgG1 and IgG2. IVIg preparations contained relatively high levels of galactosylated and sialylated IgG Fc glycoforms compared with serum IgG in patients. Treatment with IVIg resulted in an increase in serum of the Fc-galactosylation and -sialylation of both IgG1 and IgG2. The extent of normalization in serum IgG Fc glycosylation varied between patients. Multiple logistic regression analysis showed that patients with persistent low IgG galactosylation and sialylation despite IVIg treatment had the most severe forms of GBS and needed ventilator support more often. Kaplan-Meier analysis showed that these patients also needed more time to be able to walk again compared with patients with a normalized IgG Fc glycosylation profile. In conclusion, our results suggest that serum IgG Fc glycosylation in GBS is related to disease severity and clinical recovery after IVIg and may help to develop new measures to monitor the efficacy of treatment.

Sialylation of Campylobacter jejuni Endotoxin Promotes Dendritic Cell–Mediated B Cell Responses through CD14-Dependent Production of IFN-β and TNF-α

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis and often precedes development of Guillain–Barré syndrome (GBS), a life-threatening paralytic disease. The incorporation of the carbohydrate sialic acid into C. jejuni lipooligosaccharides (LOS) is associated with increased severity of gastroenteritis and with induction of GBS; however, the underlying mechanisms remain completely unknown. In this study, we demonstrate that sialic acids in C. jejuni endotoxin enhance the rapid production of IFN-β and TNF-α by human dendritic cells (DCs). Using neutralizing Abs and receptors it was shown that these DC-derived cytokines promote the proliferation of human mucosal B cells in a T cell–independent manner. The production of both IFN-β and TNF-α by DCs in response to LOS requires CD14, and the amplified response of DCs to sialylated C. jejuni LOS is CD14 dependent. Together, these results indicate that sialylation of C. jejuni LOS increases DC activation and promotes subsequent B cell responses through CD14-driven production of IFN-β and TNF-α. This enhanced DC/B cell response may explain the increased pathogenicity of sialylated C. jejuni and may be key to the initiation of B cell–mediated autoimmunity in GBS.