Immune Cell Extravasation Research Articles

Targeting the Chemokines in Cardiac Repair

Chemokines are a family of chemotactic cytokines that play an essential role in leukocyte trafficking. Upregulation of both CC and CXC chemokines is a hallmark of the inflammatory and reparative response following myocardial infarction. Release of danger signals from dying cells and damaged extracellular matrix activates innate immune pathways that stimulate chemokine synthesis. Cytokineand chemokine-driven adhesive interactions between endothelial cells and leukocytes mediate extravasation of immune cells into the infarct. CXC chemokines (such as interleukin-8) are bound to glycosaminoglycans on the endothelial surface and activate captured neutrophils, inducing expression of integrins. CC chemokines (such as monocyte chemoattractant protein (MCP)-1) mediate recruitment of proinflammatory and phagocytotic mononuclear cells into the infarct. CC Chemokines may also regulate late infiltration of the healing infarct with inhibitory leukocytes that suppress inflammation and restrain the post-infarction immune response. Non-hematopoietic cells are also targeted by chemokines; in healing infarcts, the CXC chemokine Interferon-γ inducible Protein (IP)-10 exerts antifibrotic actions, inhibiting fibroblast migration. Another member of the CXC subfamily, Stromal cell-derived Factor (SDF)-1, may protect the infarcted heart by activating pro-survival signaling in cardiomyocytes, while exerting angiogenic actions through chemotaxis of endothelial progenitors. Several members of the chemokine family may be promising therapeutic targets to attenuate adverse remodeling in patients with myocardial infarction.

Janus kinases 1 and 2 regulate chemokine‐mediated integrin activation and naïve T‐cell homing

Janus kinases (JAKs) are central signaling molecules in cytokine receptor cascades. Although they have also been implicated in chemokine receptor signaling, this function continues to be debated. To address this issue, we established a nucleofection model in primary, nonactivated mouse T lymphocytes to silence JAK expression and to evaluate the ability of these cells to home to lymph nodes. Reduced JAK1 and JAK2 expression impaired naïve T-cell migration in response to gradients of the chemokines CXCL12 and CCL21. In vivo homing of JAK1/JAK2-deficient cells to lymph nodes decreased, whereas intranodal localization and motility were unaffected. JAK1 and JAK2 defects altered CXCL12- and CCL21-triggered ezrin/radixin/moesin (ERM) dephosphorylation and F-actin polymerization, as well as activation of lymphocyte function-associated Ag-1 and very late Ag-4 integrins. As a result, the cells did not adhere firmly to integrin substrates in response to these chemokines. The results demonstrate that JAK1/JAK2 participate in chemokine-induced integrin activation and might be considered a target for modulation of immune cell extravasation and therefore, control of inflammatory reactions.

A specific subset of RabGTPases controls cell surface exposure of MT1-MMP, extracellular matrix degradation and 3D invasion of macrophages

The matrix metalloproteinase MT1-MMP has a major impact on invasive cell migration in both physiological and pathological settings such as immune cell extravasation or metastasis of cancer cells. Surface-associated MT1-MMP is able to cleave components of the extracellular matrix, which is a prerequisite for proteolytic invasive migration. However, current knowledge on the molecular mechanisms that regulate MT1-MMP trafficking to and from the cell surface is limited. We have identified three members of the RabGTPase family, Rab5a, Rab8a and Rab14, as crucial regulators of MT1-MMP trafficking and function in primary human macrophages. Both overexpressed and endogenous forms show prominent colocalisation with MT1-MMP-positive vesicles, whereas expression of mutant constructs, as well as siRNA-induced knockdown, reveal that these RabGTPases are crucial in the regulation of MT1-MMP surface exposure, contact of MT1-MMP-positive vesicles with podosomes, extracellular matrix degradation in two and three dimensions, as well as three-dimensional proteolytic invasion of macrophages. Collectively, our results identify Rab5a, Rab8a and Rab14 as major regulators of MT1-MMP trafficking and invasive migration of primary human macrophages, which could be promising potential targets for manipulation of immune cell invasion.

Improving Drug Uptake and Penetration into Tumors: Current and Forthcoming Opportunities

Improving Drug Uptake and Penetration into Tumors: Current and Forthcoming Opportunities

Inhibition of Activated Protein C Attenuated Experimental Autoimmune Encephalomyelitis (171.13)

Abstract Activated Protein C (APC) is an anticoagulant that is involved in the complex two-way interactions between the coagulation and immune systems. APC is widely known to be anti-inflammatory; most notably it has been clinically developed for treatment of severe sepsis. The anti-inflammatory properties of APC include conferring endothelial barrier protection, thus limiting the extravasation of immune cells into tissues. We investigated whether APC can affect the progression of experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis. The major pathological component of EAE is blood brain barrier (BBB) dysfunction resulting in the extravasation of autoreactive immune cells into the central nervous system (CNS). We observed that inhibition of endogenous APC during EAE resulted in considerable immune cell infiltration in the brain. Interestingly, however, these mice exhibited mild to no outward signs of EAE as assessed by paralysis severity. The anti-APC treated mice also had significantly higher percentage of CD4+Foxp3+ cells both in the CNS and in the periphery compared to controls. Moreover, the frequency of cells characterized as myeloid derived suppressor cells are increased in anti-APC treated EAE mice. Our study shows that lowered APC levels during EAE can lead to divergent effects on EAE pathogenesis, specifically leading to increased BBB dysfunction while simultaneously inducing immunological conditions that are non-pathogenic in EAE.

Tissue-Specific Homing of Immune Cells in Malignant Skin Tumors

Tissue-specific migration of immune cells involved both in physiological and pathological immune responses is a current research subject for medical science. Several homing molecules have been identified orchestrating extravasation of immune cells to certain peripheral non-lymphoid tissues such as gut, lung and skin. Regarding lymphocyte homing to skin, the first-line defense of human body cutaneous lymphocyte associated antigen (CLA) and a group of chemokine-chemokine receptor pairs are considered to be of crucial importance. The aim of the present review is to summarize existing knowledge about skin- and tumor-specific migration of immune cells playing a major pathogenetic role in host immune responses induced by non-lymphoid malignant skin tumors as well as in the development of primary cutaneous T-cell lymphomas (CTCL). Melanoma malignum, squamous and basal cell carcinoma evoke host immune responses and consequently a subset of reactive immune cells is recruited to site of the tumor. Regarding migratory process and exact functional role of these cells a growing number of data is available in literature. On the other hand tissue-specific immune cell homing is regarded as a key process in the pathogenesis of CTCL where malignant T-lymphocytes can be found in circulation and symptomatic skin. Hereby homing mechanism of malignant T-cells in mycosis fungoides and Sézary-syndrome as separate clinical entities of CTCL is discussed. A precise insight into the molecular background of skin- and tumor-specific immune cell migration can contribute to developing efficient vaccine therapies in non-lymphoid malignant skin tumors and beneficial treatment modalities in CTCL.

Natalizumab Treatment Effects Sustained Decrease in Serum Levels of Fibronectin and Soluble Adhesion Molecules sVCAM-1 and sICAM-3 (P02.114)

Objective: To obtain a better understanding of potential additional effector mechanisms of natalizumab we analyzed the influence of short and long-term treatment on soluble adhesion molecules (sAMs) in peripheral blood of MS patients. Background Natalizumab (Tysabri) is a humanized monoclonal antibody used in the treatment of relapsing-remitting multiple sclerosis (MS). It directly binds to the alpha4-integrin subunit of very late activation antigen-4 (VLA-4) and results in decreased detectability of unblocked alpha-4 leading to an inhibition of immune cell extravasation across the blood-brain-barrier. The impressive therapeutic effect of natalizumab is overshadowed by rare but potentially serious side effects such as progressive multifocal leukoencephalopathy. Design/Methods: Serum levels of fibronectin (FN) and 4 different sAMs (soluble intercellular adhesion molecule-1,-2,-3 (sICAM-1,-2,-3) and vascular cell adhesion molecule-1 (sVCAM-1)) were analyzed before (baseline, n=16), 3 months after (short-term, n=16) and 24 months after (long-term, n=14) onset of Natalizumab treatment by fluorescent bead immunoassay and enzyme-linked immunosorbent assay. Results: A significant and sustained decrease in serum concentrations of FN, sICAM-3 and sVCAM-1 was observed during the first 3 months (FN p Conclusions: The observed decreases in serum levels highlight sICAM-3, sVCAM-1, and FN to be affected by natalizumab therapy. Decreases in sVCAM-1 serum levels have already been previously reported. We add sICAM-3 and the soluble form of the extracellular matrix protein FN. Both, sVCAM-1 and FN have been shown to be directly engaged in VLA-4-mediated extravasation of immune cells and sICAM-3 is important in the initiation of immune responses. Decreased serum levels indicate potential therapy-induced interference with fine-tuning and/or feedback mechanisms between endothelium and immune cells with yet unresolved consequences. Supported by: Biogen-Idec Austria. Disclosure: Dr. Kraus has received personal compensation for activities with Biogen Idec, Bayer Pharmaceuticals Corporation, Sanofi-Aventis Pharmaceuticals, Merck-Serono, Novartis, Genzyme Corporation as a consultant. Dr. Kraus has received research support from Biogen Idec, Bayer Pharmaceuticals Corporation, Sanofi-Aventis Pharmaceuticals, Merck Serono, Novartis, Genzyme Corporation. Dr. Oppermann has nothing to disclose. Dr. Pilz has nothing to disclose. Dr. Wipfler has nothing to disclose. Dr. Afazel has nothing to disclose. Dr. Haschke-Becher has nothing to disclose. Dr. Trinka has received personal compensation for activities with UCB Pharma, Eisai Inc., Sunovion, Medtronic, Pfizer Inc, Ever-Neuropharma and GlaxoSmithKline, Inc. as a consultant and/or speaker. Dr. Trinka has received research support from UCB Pharma, Ever-Neuropharma, Medtronic and GlaxoSmithKline, Inc. Dr. Harrer has nothing to disclose.

Role of cold shock Y-box protein-1 in inflammation, atherosclerosis and organ transplant rejection

Chemokines (chemoattractant cytokines) are crucial regulators of immune cell extravasation from the bloodstream into inflamed tissue. Dysfunctional regulation and perpetuated chemokine gene expression are linked to progressive chronic inflammatory diseases and, in respect to transplanted organs, may trigger graft rejection. RANTES (regulated upon activation, normal T cell expressed and secreted (also known as CCL5)) is a model chemokine with relevance in numerous inflammatory diseases where the innate immune response predominates. Transcription factor Y-box binding protein-1 (YB-1) serves as a trans-regulator of CCL5 gene transcription in vascular smooth muscle cells and leucocytes. This review provides an update on YB-1 as a mediator of inflammatory processes and focuses on the role of YB-1 in CCL5 expression in diseases with monocytic cell infiltrates, albeit acute or chronic. Paradigms of such diseases encompass atherosclerosis and transplant rejection where cold shock protein YB-1 takes a dominant role in transcriptional regulation.

Inhibition of activated protein C dramatically attenuated experimental autoimmune encephalomyelitis (164.9)

Abstract Activated Protein C (APC) is an anticoagulant that is involved in the complex two-way interactions between the coagulation and immune systems. APC is widely known to be anti-inflammatory; most notably it has been clinically developed for treatment of severe sepsis. The anti-inflammatory properties of APC include conferring endothelial barrier protection, thus limiting the extravasation of immune cells into tissues. Given that the major pathological component of multiple sclerosis and its mouse model, experimental autoimmune encephalomyelitis (EAE), is the extravasation of autoreactive immune cells in the central nervous system (CNS) due to blood brain barrier (BBB) dysfunction, we investigated whether APC can affect the progression of EAE. We saw that blocking endogenous APC during EAE resulted in considerable immune cell infiltration in the CNS. Interestingly, however, these mice exhibited mild to no outward signs of EAE as assessed by paralysis severity. The anti-APC treated mice also had significantly higher percentage of CD4+Foxp3+ cells both in the CNS and in the periphery compared to controls. Moreover, the cytokine profile of leukocytes from the anti-APC mice was skewed to T helper-2, which is known to be protective in EAE. These data show that lowered APC levels during EAE can lead to divergent effects on EAE pathogenesis, specifically leading to increased BBB dysfunction while simultaneously inducing immunological conditions that are non-pathogenic in EAE.

Adhesion molecules are promising candidates to establish surrogate markers for natalizumab treatment

Background: Natalizumab is the first monoclonal antibody therapy approved for multiple sclerosis (MS). Its therapeutic mechanism is the blockade of the α4-integrin subunit of the adhesion molecule (AM) very late activation antigen-4 (VLA-4), which leads to an inhibition of immune cell extravasation into the central nervous system (CNS). Methods: We investigated changes in the expression levels of unblocked α4-integrin and further AM (intercellular adhesion molecule-1, -2, -3 (cICAM-1, -2, -3), leukocyte function associated antigen-1 (LFA-1)) on peripheral blood mononuclear cells (PBMC) determined by flow cytometry from 25 patients with MS before the first natalizumab infusion and before the fourth infusion. In 15 MS patients AM expression was evaluated every 3 months over 1 year. Results:We found a significant decrease (p < 0.0001) of unblocked α4-integrin cell surface expression on all investigated PBMC subsets (T cells −61.7%, B cells −69.1%, monocytes/macrophages −46.4%) in the blood of MS patients after 3 months of natalizumab treatment. Moreover, a continuous decrease (p < 0.05) of unblocked α4-integrin expression levels was seen after 3, 6, 9, and 12 months. As a secondary effect, expression levels of the other investigated AM were differentially affected. Conclusions:Results show a sustained decrease of unblocked α4-integrin expression not only in all patients but also in all investigated PBMC subsets. This probably results in a continuously decreasing transmigration of PBMC into the CNS and may explain the improved clinical efficacy in the second treatment year and also the increasing risk of progressive multifocal leukoencephalopathy during long-term natalizumab therapy. We conclude that AM expression profiles are promising candidates for the development of a biomarker system to determine both natalizumab treatment response and patients at risk for opportunistic CNS infections.

Neurogenic inflammation of the ocular surface

The purpose of the present review is to describe the involvement of neurogenic inflammation in ocular surface diseases and to outline recent advances in understanding the complex interaction of neuromediators and inflammatory cells in the conjunctiva. Evidence indicates that all clinical forms of ocular surface disease, including allergic and autoimmune, share the involvement of a neurogenic inflammation triggered by nociceptive stimuli and resulting in plasma extravasation and local activation of immune cells. Players of this complex mechanism are neuropeptides, including substance P, calcitonine gene-related peptide, neuropeptide Y, and vasoactive intestinal peptide. These neuropeptides are, however, only a minor component of a complex neuroimmune cross-talk capable of modulating local inflammation. As already shown in other mucosal surfaces, neurogenic inflammation and innate immunity may work together to protect the ocular surface. In this review, we present the most recent advances in the understanding of the roles played by nerve endings and neuropeptides in local inflammatory processes of the ocular surface, in order to better clarify their function in physiologic and pathologic conditions and to open new research, diagnostic, and therapeutic perspectives.

Cytokines and chemokines in testicular inflammation: A brief review

A wide spectrum of data in the literature shows the relevance of cytokines as paracrine regulators of spermatogenesis and steroidogenesis in the normal testis. In this brief review, we highlight the relevance of cytokines in the testis during inflammation. This phenomenon involves complex and multiple interactions among immune and germ cells generally resulting in the alteration of spermatogenesis. The complexity of these cell interactions is multiplied because Sertoli and Leydig cells are also producers of pro- and anti-inflammatory cytokines and chemokines. Also, cytokines are pleiotropic and they exert opposite and/or redundant effects in different conditions. However, in spite of this bidirectional immunoregulatory function of cytokines, the mass of the data, reported from experiments of acute testicular inflammation, shows upregulation of interleukin (IL)-1beta, IL-1alpha, IL-6, and tumor necrosis factor alpha (TNF-alpha), which induce adverse effects on germ cells. In autoimmune orchitis, a chronic testicular inflammation, chemokines such as CCL2, CCL3, and CCL4 induce attraction and extravasation of immune cells within the testicular interstitium. These cells alter the normal immunosuppressor microenvironment principally through the secretion of proinflammatory cytokines, interferon-gamma initially, and IL-6 and TNF-alpha thereafter. Germ cells expressing TNFR1, IL-6R, and Fas increase in number and undergo apoptosis, through the TNF-alpha/TNFR1, IL-6/IL-6R, and Fas/Fas L systems. The knowledge of immune-germ and somatic testicular cell interactions will contribute to the understanding of the mechanisms by which chronic inflammatory conditions of the testis can disrupt the process of spermatogenesis.

Die Rolle von Endothelzellen in der Transplantatabstoßung

Endothelial cells (EC) are crucially involved in allograft rejection. They are prime targets of alloreactivity but also key players in the recruitment and extravasation of immune cells. These mechanisms also become clear in allograft biopsies with antibody-mediated complement deposition on EC and associated intracapillary accumulation of immune cells. HLA molecules are the most prominent targets of alloantibodies in AB0 compatible transplantation. Clinically relevant antibodies against other antigens such as MICA (MHC class I-related chain A) or the angiotensin II Type-1 receptor could also be convincingly demonstrated. The lack of generally available diagnostic tests for such non-HLA antibodies hampers their introduction into clinical practice. Alloantibodies undoubtedly cause allograft rejection. However, our knowledge of the molecular mechanisms underlying graft dysfunction in antibody-mediated rejection (AMR) is still fragmentary. Activation of EC by anti-endothelial cell antibodies was demonstrated in several experimental systems. Recent animal studies employing immune cell deficient transplant recipients or in-vitro assays, however, failed to demonstrate an immediate response of EC upon antibody binding and complement activation. It might therefore be considered that direct antibody- or complement-mediated EC damage is not necessarily the leading event in acute AMR. Antibody- and/or complement-induced recruitment of immune cells might rather be of crucial importance at least in the early phases of AMR.

All-trans-Retinoic Acid Induces Integrin-Independent B-Cell Adhesion to ADAM Disintegrin Domains

Cell adhesion is an integral aspect of immunity facilitating extravasation of immune cells during homing and activation. All -trans-Retinoic acid ( t-RA) regulates leukocyte differentiation, proliferation, and transmigration. However, the role of t-RA in immune cell adhesion is poorly defined. In this study, we evaluated the impact of t-RA and its metabolism on B and T cell adhesion. Specifically, we address the impact of t-RA on the adhesive properties of the human mature B and T cell lines RPMI 8866, Daudi and Jurkats. The effect of t-RA exposure on cell adhesion to vascular cell adhesion molecule-1 (VCAM-1), a well-established integrin counter receptor involved in immunity, and to nonconventional ADAM integrin ligands was assessed. We show for the first time that t-RA potently induces B cell adhesion in an integrin-independent manner to both VCAM-1 and select ADAM disintegrin domains. Using retinoid extraction and reverse-phase HPLC analysis, we identify the retinoid that is functionally responsible for this augmented adhesion. We also provide evidence that this novel t-RA adhesive response is not prototypical of lymphocytes since both Daudi and Jurkats do not alter their adhesive properties upon t-RA treatment. Further, the t-RA metabolic profiles between these lineages is distinct with 9- cis-retinoic acid being exclusively detected in Jurkat media. This study is the first to demonstrate that t-RA directly induces B cell adhesion in an integrin-independent manner and is not contingent upon t-RA metabolism.

Involvement of heparanase in migration of microglial cells

Heparanase, a matrix-degrading enzyme that cleaves heparan sulfate side chains from heparan sulfate proteoglycans (HSPGs), has been shown to facilitate cell invasion, migration, and extravasation of metastatic tumor cells or immune cells. In this study, the expression and functions of heparanase were investigated using rat primary cultured microglia, the resident macrophages in the brain. The microglia were found to express heparanase mRNA and protein. Microglia treated with lipopolysaccharide (LPS) were activated, expressed induced nitric oxide synthase and elevated the expression of heparanase. Heparanase has two molecular weights: a 65 kDa latent form and an active 50 kDa. Both forms were expressed by LPS-treated activated microglia; however, untreated microglia primarily expressed the latent form. Cell lysates from microglia actually degraded Matrigel containing HSPG. Heparanase was colocalized with the actin cytoskeleton in microglial leading edges or ruffled membranes. Microglia transmigrated through a Matrigel-coated pored membrane. This process was inhibited by SF-4, a specific heparanase inhibitor, in a concentration-dependent manner. Degraded HSPG was generated when microglia transmigrated through the coated membrane, and this was also inhibited by SF-4. The results suggest the involvement of heparanase in the migration or invasion of microglia or brain macrophages across basement membrane around brain vasculature.

Role of endothelial heparanase in delayed-type hypersensitivity

Heparanase is an endoglycosidase that cleaves heparan sulfate (HS), the main polysaccharide of the basement membrane (BM). HS is responsible for BM integrity and barrier function. Hence, enzymatic degradation of HS in the vascular subendothelial BM is a prerequisite for extravasation of immune cells and plasma components during inflammation. Here, we demonstrate a highly coordinated local heparanase induction upon elicitation of delayed-type hypersensitivity (DTH) reaction in the mouse ear. By monitoring in vivo activation of luciferase gene driven by the heparanase promoter, we demonstrate activation of heparanase transcription at an early stage of DTH. We report that heparanase is produced locally by the endothelium at the site of DTH-associated inflammation. Key DTH mediators, tumor necrosis factor-alpha and interferon-gamma, were found to induce heparanase in cultured endothelial cells. Endothelium emerges as an essential cellular source of heparanase enzymatic activity that, in turn, allows for remodeling of the vascular BM, increased vessel permeability, and extravasation of leukocytes and plasma proteins. In vivo administration of antiheparanase siRNA or an inhibitor of heparanase enzymatic activity effectively halted DTH inflammatory response. Collectively, our results highlight the decisive role of endothelial heparanase in DTH inflammation and its potential as a promising target for anti-inflammatory drug development.

Subcutaneous Interferon-β injections in patients with multiple sclerosis initiate inflammatory skin reactions by local chemokine induction

Subcutaneous Interferon-β (IFN-β) injections for the treatment of multiple sclerosis (MS) frequently cause inflammatory injection site reactions. To study the role of chemokines we obtained skin biopsies from 7 MS patients 24 h after injection. At the IFN-β but not at the contralateral placebo injection sites, we observed strong IP-10/CXCL10 and moderate MCP-1/CCL2 expression associated with extensive perivascular, highly CXCR3-positive T cell and macrophage infiltrates. Primary human skin cells displayed a comparable pattern of chemokine induction after stimulation with IFN-β in vitro. IFN-β may therefore trigger inflammatory skin reactions through local chemokine induction followed by rapid immune cell extravasation.

The mechanism of action of methylprednisolone in the treatment of multiple sclerosis

Methylprednisolone plays an important role in the current treatment of multiple sclerosis (MS), particularly in the acute phase of relapse. It acts in various ways to decrease the inflammatory cycle including: dampening the inflammatory cytokine cascade, inhibiting the activation of T cells, decreasing the extravasation of immune cells into the central nervous system, facilitating the apoptosis of activated immune cells, and indirectly decreasing the cytotoxic effects of nitric oxide and tumor necrosis factor alpha. This paper reviews the most recent observations on these mechanisms both to understand the disease mechanism and its treatment. As more becomes known about these mechanisms, it may become possible to design treatment regimes that are more specific towards both the individual and the disease state.

The effect of topical radiant heating on local blood flow and immune cell extravasation in split‐thickness skin graft donor site wounds

The effect of topical radiant heating on local blood flow and immune cell extravasation in split‐thickness skin graft donor site wounds

Brain resistance to HSV-1 encephalitis in a mouse model

Brain resistance to intracerebral superinfections develops after a peripheral inoculation of neurovirulent viruses. Superinfection resistance combines specificity, toward the virus used for the peripheral inoculum, and short-term duration after the inoculum. In order to study this unusual combination, neurovirulent superinfections were made on albino Swiss mice previously infected with a nasal inoculum. A herpesvirus strain SC16, or a homologue recombinant virus carrying the reporter lac Z gene or a vesicular stomatitis virus (VSV) (a virus taxonomically unrelated to Herpesviridae) were used. The mice underwent a neurological examination and their survival rate was recorded. The brains superinfected with the reporter virus were stained for the beta-galactosidase reaction to trace the virus spread and the inflammatory infiltrates were characterized immunocytochemically. The results confirm and extend previous observations about virus specificity and short-term duration of superinfection resistance. They show, moreover, an enhanced brain inflammation with T-cells and macrophages infiltrating the tissue around microvessels, at a time when both neurovirulence and the spread of herpesvirus in the brain are reduced. The results suggest that the immune response to superinfection in the nervous tissue is enhanced by blood-brain barrier mechanisms that promote the timely extravasation of immune cells.