Liora Cahalon

Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix

On their extravasation from the vascular system into inflamed tissues, leukocytes must maneuver through a complex insoluble network of molecules termed the extracellular matrix (ECM). Leukocytes navigate toward their target sites by adhering to ECM glycoproteins and secreting degradative enzymes, while constantly orienting themselves in response to specific signals in their surroundings. Cytokines and chemokines are key biological mediators that provide such signals for cell navigation. Although the individual effects of various cytokines have been well characterized, it is becoming increasingly evident that the mixture of cytokines encountered in the ECM provides important combinatorial signals that influence cell behavior. Herein, we present an overview of previous and ongoing studies that have examined how leukocytes integrate signals from different combinations of cytokines that they encounter either simultaneously or sequentially within the ECM, to dynamically alter their navigational activities. For example, we describe our findings that tumor necrosis factor (TNF)‐α acts as an adhesion‐strengthening and stop signal for T cells migrating toward stromal cell‐derived factor‐1α, while transforming growth factor‐β down‐regulates TNF‐α‐induced matrix metalloproteinase‐9 secretion by monocytes. These findings indicate the importance of how one cytokine, such as TNF‐α, can transmit diverse signals to different subsets of leukocytes, depending on its combination with other cytokines, its concentration, and its time and sequence of exposure. The combinatorial effects of multiple cytokines thus affect leukocytes in a step‐by‐step manner, whereby cells react to cytokine signals in their immediate vicinity by altering their adhesiveness, directional movement, and remodeling of the ECM.

CNS-specific immunity at the choroid plexus shifts toward destructive Th2 inflammation in brain aging

The adaptive arm of the immune system has been suggested as an important factor in brain function. However, given the fact that interactions of neurons or glial cells with T lymphocytes rarely occur within the healthy CNS parenchyma, the underlying mechanism is still a mystery. Here we found that at the interface between the brain and blood circulation, the epithelial layers of the choroid plexus (CP) are constitutively populated with CD4+ effector memory cells with a T-cell receptor repertoire specific to CNS antigens. With age, whereas CNS specificity in this compartment was largely maintained, the cytokine balance shifted in favor of the T helper type 2 (Th2) response; the Th2-derived cytokine IL-4 was elevated in the CP of old mice, relative to IFN-γ, which decreased. We found this local cytokine shift to critically affect the CP epithelium, triggering it to produce the chemokine CCL11 shown to be associated with cognitive dysfunction. Partial restoration of cognitive ability in aged mice, by lymphopenia-induced homeostasis-driven proliferation of memory T cells, was correlated with restoration of the IL-4:IFN-γ ratio at the CP and modulated the expression of plasticity-related genes at the hippocampus. Our data indicate that the cytokine milieu at the CP epithelium is affected by peripheral immunosenescence, with detrimental consequences to the aged brain. Amenable to immunomodulation, this interface is a unique target for arresting age-related cognitive decline.

Dopamine interacts directly with its D3 and D2 receptors on normal human T cells, and activates β1 integrin function

Dopamine by itself has not up to now been reported to activate T cell function. We show here that dopamine interacts directly with dopaminergic receptors on normal human T cells and triggers β1 integrin‐mediated T cell adhesion to a major extracellular matrix component, fibronectin (FN). Such adhesion is a characteristic feature of activated T cells, and is critical for trafficking and extravasation of T cells across blood vessels and tissue barriers. Seven dopamine D2/D3 receptor agonists and antagonists were used to identify the receptor subtypes with which dopamine specifically interacts to activate T cells. The D3 dopamine receptor agonist, 7‐hydroxy‐DPAT (DPAT), mimics the effects of dopamine, and the effects of both dopamine and DPAT are blocked by a specific D3 receptor antagonist, U‐maleate. The dopamine receptor agonists bromocriptine and pergolide mimic the direct effect of dopamine on the β1 integrin function, while the dopamine receptor antagonists butaclamol and haloperidol suppress it, suggesting additional signaling via the dopamine D2 receptor subtype. Our study shows, for the first time, that dopamine can directly activate T cells via ist specific receptors and suggests a possible role for dopamine in integrin‐mediated cellular trafficking and extravasation of T cells in the central nervous system and possibly also in the periphery. Finally, we suggest that the reported changes in the D3 and D2 receptor RNA levels in peripheral blood lymphocytes of individuals with schizophrenia, Parkinsons disease, Alzheimers disease and migraine can serve not only as a ‘passive’ diagnostic marker, but primarily reflect the dynamic functional dopamine‐T cell interactions in these diseases.

Breaking immune tolerance by targeting Foxp3(+) regulatory T cells mitigates Alzheimer's disease pathology.

Alzheimers disease (AD) is a neurodegenerative disorder in which chronic neuroinflammation contributes to disease escalation. Nevertheless, while immunosuppressive drugs have repeatedly failed in treating this disease, recruitment of myeloid cells to the CNS was shown to play a reparative role in animal models. Here we show, using the 5XFAD AD mouse model, that transient depletion of Foxp3+ regulatory T cells (Tregs), or pharmacological inhibition of their activity, is followed by amyloid-β plaque clearance, mitigation of the neuroinflammatory response and reversal of cognitive decline. We further show that transient Treg depletion affects the brains choroid plexus, a selective gateway for immune cell trafficking to the CNS, and is associated with subsequent recruitment of immunoregulatory cells, including monocyte-derived macrophages and Tregs, to cerebral sites of plaque pathology. Our findings suggest targeting Treg-mediated systemic immunosuppression for treating AD.

Somatostatin Through Its Specific Receptor Inhibits Spontaneous and TNF-α- and Bacteria-Induced IL-8 and IL-1β Secretion from Intestinal Epithelial Cells

Intestinal epithelial cells secrete proinflammatory cytokines and chemokines that are crucial in mucosal defense. However, this secretion must be tightly regulated, because uncontrolled secretion of proinflammatory mediators may lead to chronic inflammation and mucosal damage. The aim of this study was to determine whether somatostatin, secreted within the intestinal mucosa, regulates secretion of cytokines from intestinal epithelial cells. The spontaneous as well as TNF-α- and Salmonella-induced secretion of IL-8 and IL-1β derived from intestinal cell lines Caco-2 and HT-29 was measured after treatment with somatostatin or its synthetic analogue, octreotide. Somatostatin, at physiological nanomolar concentrations, markedly inhibited the spontaneous and TNF-α-induced secretion of IL-8 and IL-1β. This inhibition was dose dependent, reaching >90% blockage at 3 nM. Furthermore, somatostatin completely abrogated the increased secretion of IL-8 and IL-1β after invasion by Salmonella. Octreotide, which mainly stimulates somatostatin receptor subtypes 2 and 5, affected the secretion of IL-8 and IL-1β similarly, and the somatostatin antagonist cyclo-somatostatin completely blocked the somatostatin- and octreotide-induced inhibitory effects. This inhibition was correlated to a reduction of the mRNA concentrations of IL-8 and IL-1β. No effect was noted regarding cell viability. These results indicate that somatostatin, by directly interacting with its specific receptors that are expressed on intestinal epithelial cells, down-regulates proinflammatory mediator secretion by a mechanism involving the regulation of transcription. These findings suggest that somatostatin plays an active role in regulating the mucosal inflammatory response of intestinal epithelial cells after physiological and pathophysiological stimulations such as bacterial invasion.

Cutting edge: T cells respond to lipopolysaccharide innately via TLR4 signaling.

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1α (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.

A sulfated disaccharide derived from chondroitin sulfate proteoglycan protects against inflammation-associated neurodegeneration

Chondroitin sulfate proteoglycan (CSPG), a matrix protein that occurs naturally in the central nervous system (CNS), is considered to be a major inhibitor of axonal regeneration and is known to participate in activation of the inflammatory response. The degradation of CSPG by a specific enzyme, chondroitinase ABC, promotes repair. We postulated that a disaccharidic degradation product of this glycoprotein (CSPG‐DS), generated following such degradation, participates in the modulation of the inflammatory responses and can, therefore, promote recovery in immune‐induced neuropathologies of the CNS, such as experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU). In these pathologies, the dramatic increase in T cells infiltrating the CNS is far in excess of the numbers needed for regular maintenance. Here, we show that CSPG‐DS markedly alleviated the clinical symptoms of EAE and protected against the neuronal loss in EAU. The last effect was associated with a reduction in the numbers of infiltrating T cells and marked microglia activation. This is further supported by our in vitro results indicating that CSPG‐DS attenuated T cell motility and decreased secretion of the cytokines interferon‐? and tumor necrosis factor‐?. Mechanistically, these effects are associated with an increase in SOCS‐3 levels and a decrease in NF‐?B. Our results point to a potential therapeutic modality, in which a compound derived from an endogenous CNS‐resident molecule, known for its destructive role in CNS recovery, might be helpful in overcoming inflammation‐induced neurodegenerative conditions.

Enzymatically Quiescent Heparanase Augments T Cell Interactions with VCAM-1 and Extracellular Matrix Components under Versatile Dynamic Contexts

During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1α (SDF-1α; CXCL12), in an α4β1-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in β1 integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells’ surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1α-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1α-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites.

A disaccharide derived from chondroitin sulphate proteoglycan promotes central nervous system repair in rats and mice

Chondroitin sulphate proteoglycan (CSPG) inhibits axonal regeneration in the central nervous system (CNS) and its local degradation promotes repair. We postulated that the enzymatic degradation of CSPG generates reparative products. Here we show that an enzymatic degradation product of CSPG, a specific disaccharide (CSPG‐DS), promoted CNS recovery by modulating both neuronal and microglial behaviour. In neurons, acting via a mechanism that involves the PKCα and PYK2 intracellular signalling pathways, CSPG‐DS induced neurite outgrowth and protected against neuronal toxicity and axonal collapse in vitro. In microglia, via a mechanism that involves ERK1/2 and PYK2, CSPG‐DS evoked a response that allowed these cells to manifest a neuroprotective phenotype ex vivo. In vivo, systemically or locally injected CSPG‐DS protected neurons in mice subjected to glutamate or aggregated β‐amyloid intoxication. Our results suggest that treatment with CSPG‐DS might provide a way to promote post‐traumatic recovery, via multiple cellular targets.

Fibronectin‐bound TNF‐α stimulates monocyte matrix metalloproteinase‐9 expression and regulates chemotaxis

Tumor necrosis factor α (TNF‐α) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF‐α avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF‐α complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM‐bound TNF‐α by examining its effects on MMP‐9 secretion by monocytes. Immunofluorescent staining indicated that LPS‐activated monocytes deposited newly synthesized TNF‐α into ECM‐FN. FN‐bound TNF‐α (FN/TNF‐α) significantly up‐regulated MMP‐9 expression and secretion by the human monocytic cell line MonoMac‐6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF‐α activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Chemotaxis through ECM gels in the presence of soluble or bound TNF‐α was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac‐6 cells to FN/TNF‐α required functional activated β1 integrins, FN/TNF‐α‐induced MMP‐9 secretion was independent of binding to β1 integrins, since MMP‐9 secretion was unaffected by: (1) neutralizing mAb to α4, α5, and β1 subunits, which blocked cell adhesion; (2) a mAb that stimulated β1 integrin‐mediated adhesion; and (3) binding TNF‐α to the 30‐kDa amino‐terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell‐adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM‐degrading enzymes. These TNF‐α‐ECM interactions may serve to limit the cytokine’s availability and bioactivity to target areas of inflammation.