Marika Karikoski

Altered purinergic signaling in CD73 deficient mice inhibits tumor progression

CD73/ecto‐5′‐nucleotidase dephosphorylates extracellular AMP into adenosine, and it is a key enzyme in the regulation of adenosinergic signaling. The contribution of host CD73 to tumor growth and anti‐tumor immunity has not been studied. Here, we show that under physiological conditions CD73‐deficient mice had significantly elevated ATPase and ADPase activities in LN T cells. In a melanoma model, the growth of primary tumors and formation of metastasis were significantly attenuated in mice lacking CD73. Among tumor‐infiltrating leukocytes there were fewer Tregs and mannose receptor‐positive macrophages, and increased IFN‐γ and NOS2 mRNA production in CD73‐deficient mice. Treatment of tumor‐bearing animals with soluble apyrase, an enzyme hydrolyzing ATP and ADP, significantly inhibited tumor growth and accumulation of intratumoral Tregs and mannose receptor‐positive macrophages in the WT C57BL/6 mice but not in the CD73‐deficient mice. Pharmacological inhibition of CD73 with α,β‐methylene‐adenosine‐5′‐diphosphate in WT mice retarded tumor progression similarly to the genetic deletion of CD73. Together these data show that increased pericellular ATP degradation in the absence of CD73 activity in the host cells is a novel mechanism controlling anti‐tumor immunity and tumor progression, and that the purinergic balance can be manipulated therapeutically to inhibit tumor growth.

Regulation of mucosal addressin cell adhesion molecule 1 expression in human and mice by vascular adhesion protein 1 amine oxidase activity

Primary sclerosing cholangitis (PSC) and autoimmune hepatitis are hepatic complications associated with inflammatory bowel disease (IBD). The expression of mucosal addressin cell adhesion molecule 1 (MAdCAM‐1) on mucosal endothelium is a prerequisite for the development of IBD, and it is also detected on the hepatic vessels of patients with liver diseases associated with IBD. This aberrant hepatic expression of MAdCAM‐1 results in the recruitment of effector cells initially activated in the gut to the liver, in which they drive liver injury. However, the factors responsible for the aberrant hepatic expression of MAdCAM‐1 are not known. In this study, we show that deamination of methylamine (MA) by vascular adhesion protein 1 (VAP‐1) [a semicarbazide‐sensitive amine oxidase (SSAO) expressed in the human liver] in the presence of tumor necrosis factor α induces the expression of functional MAdCAM‐1 in hepatic endothelial cells and in intact human liver tissue ex vivo. This is associated with increased adhesion of lymphocytes from patients with PSC to hepatic vessels. Feeding mice MA, a constituent of food and cigarette smoke found in portal blood, led to VAP‐1/SSAO–dependent MAdCAM‐1 expression in mucosal vessels in vivo. Conclusion: Activation of VAP‐1/SSAO enzymatic activity by MA, a constituent of food and cigarette smoke, induces the expression of MAdCAM‐1 in hepatic vessels and results in the enhanced recruitment of mucosal effector lymphocytes to the liver. This could be an important mechanism underlying the hepatic complications of IBD. (HEPATOLOGY 2011;53:661‐672)

The prototype endothelial marker PAL-E is a leukocyte trafficking molecule

Pathologische Anatomie Leiden-endothelium antibody has been used for more than 20 years as a marker for vascular endothelium. Despite its widespread use, the target of this antibody was only recently identified as plasmalemma vesicle-associated protein-1 (PV-1). However, no function has been identified for this molecule. Here we report that activation of human umbilical vein endothelial cells with tumor necrosis factor-alpha resulted in a remarkable redistribution of PV-1 toward the peripheral areas of the cells. Furthermore, in vitro endpoint transmigration experiments showed that transcellularly migrating lymphocytes are surrounded by rings containing PV-1 and caveolin-1. Moreover, PV-1 associates physically with vimentin. In addition, administration of anti-PV-1 antibody during capillary flow assays resulted in a significant inhibition of lymphocyte transmigration through the endothelial cell layer, whereas rolling and adhesion were unaffected. In vivo blockage of PV-1 by an antibody in acute peritonitis and air pouch model resulted in a significant decrease in the number of migrating leukocytes. Here we thus define leukocyte transendothelial migration as the first known function for PV-1.

Erratum: The endothelial protein PLVAP in lymphatics controls the entry of lymphocytes and antigens into lymph nodes

In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens.

Clever‐1/Stabilin‐1 regulates lymphocyte migration within lymphatics and leukocyte entrance to sites of inflammation

Clever‐1/Stabilin‐1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever‐1/Stabilin‐1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever‐1/Stabilin‐1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever‐1/Stabilin‐1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti‐inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti‐Clever‐1/Stabilin‐1 treatment can target two different arms of the vasculature – traffic via lymphatics and inflamed blood vessels.

CD44 Binds to Macrophage Mannose Receptor on Lymphatic Endothelium and Supports Lymphocyte Migration via Afferent Lymphatics

Rationale: Macrophage mannose receptor (MRC) is one of the few molecules known to be involved in lymphocyte trafficking via the lymphatic vessels. In endothelial cells of efferent lymphatics, it binds L-selectin on lymphocytes. In afferent lymphatics, MRC mediates trafficking of both normal and malignant L-selectin–negative cells to the draining lymph nodes. Objective: This work was designed to search for additional lymphocyte ligands of MRC to elucidate how lymphocytes migrate into the draining lymph nodes. Methods and Results: Using immunoprecipitation and binding studies with natural and recombinant proteins, we show that MRC and CD44 can interact with each other. Fine mapping revealed that the cysteine-rich domain of MRC binds to the chondroitin sulfate side chains of CD44. In vivo homing experiments with MRC- and CD44-deficient mice verified that MRC and CD44 function as a receptor-ligand pair in supporting lymphocyte migration via the afferent lymphatics into the draining lymph nodes. Conclusions: These data identify a new counter-receptor for MRC and reveal CD44 as a new molecule involved in the poorly understood process of lymphocyte transit via the lymphatic vasculature.

SHARPIN Regulates Uropod Detachment in Migrating Lymphocytes

SHARPIN-deficient mice display a multiorgan chronic inflammatory phenotype suggestive of altered leukocyte migration. We therefore studied the role of SHARPIN in lymphocyte adhesion, polarization, and migration. We found that SHARPIN localizes to the trailing edges (uropods) of both mouse and human chemokine-activated lymphocytes migrating on intercellular adhesion molecule-1 (ICAM-1), which is one of the major endothelial ligands for migrating leukocytes. SHARPIN-deficient cells adhere better to ICAM-1 and show highly elongated tails when migrating. The increased tail lifetime in SHARPIN-deficient lymphocytes decreases the migration velocity. The adhesion, migration, and uropod defects in SHARPIN-deficient lymphocytes were rescued by reintroducing SHARPIN into the cells. Mechanistically, we show that SHARPIN interacts directly with lymphocyte-function-associated antigen-1 (LFA-1), a leukocyte counterreceptor for ICAM-1, and inhibits the expression of intermediate and high-affinity forms of LFA-1. Thus, SHARPIN controls lymphocyte migration by endogenously maintaining LFA-1 inactive to allow adjustable detachment of the uropods in polarized cells.

Clever-1/Stabilin-1 Controls Cancer Growth and Metastasis

Purpose: Immunosuppressive leukocytes and vasculature are important host cell components regulating tumor progression. Clever-1/Stabilin-1, a multifunctional scavenger and adhesion receptor, is constitutively present on a subset of type II macrophages and lymphatic endothelium, but its functional role in cancer is unknown. Experimental Design: Here, we generated full Clever-1 knockout mice and cell-specific ones lacking Clever-1 either on macrophages or endothelium. We also used anti-Clever-1 antibody therapy to treat B16 melanoma and EL-4 lymphoma. Results: Clever-1–deficient mice had smaller primary and metastatic tumors than wild-type (WT) controls. Growth of primary tumors, but not of metastases, was attenuated also in mice lacking Clever-1 selectively in macrophages or in vascular endothelium. Anti-Clever-1 antibody treatment inhibited tumor progression in WT mice. Both genetically and therapeutically induced absence of functional Clever-1 led to diminished numbers of immunosuppressive leukocyte types in tumors. Functionally Clever-1 mediated binding of immunosuppressive leukocytes to the intratumoral blood vessels aberrantly expressing Clever-1, and tumor cell traffic via the lymphatics. The antibody therapy did not aggravate autoimmunity. Conclusion: This work identifies Clever-1 in type II macrophages and in tumor vasculature as a new immunosuppressive molecule in cancer. Our finding that Clever-1 supports binding of tumor-infiltrating lymphocytes to tumor vasculature increases our understanding of leukocyte immigration to tumors. The ability of anti-Clever-1 antibody treatment to attenuate tumor progression in WT mice in vivo is therapeutically relevant. Thus, Clever-1 may be an emerging new target for modulating immune evasion and lymphatic spread in cancer. Clin Cancer Res; 20(24); 6452–64. ©2014 AACR.

VAP-1-deficient mice display defects in mucosal immunity and antimicrobial responses: implications for antiadhesive applications.

VAP-1, an ecto-enzyme expressed on the surface of endothelial cells, is involved in leukocyte trafficking between the blood and tissues under physiological and pathological conditions. In this study, we used VAP-1-deficient mice to elucidate whether absence of VAP-1 alters the immune system under normal conditions and upon immunization and microbial challenge. We found that VAP-1-deficient mice display age-dependent paucity of lymphocytes, in the Peyer’s patches of the gut. IgA concentration in serum was also found to be lower in VAP-1−/− animals than in wild-type mice. Although there were slightly less CD11a on B and T cells isolated from VAP-1-deficient mice than on those from wild-type mice, there were no differences in the expression of gut-homing-associated adhesion molecules or chemokine receptors. Because anti-VAP-1 therapies are being developed for clinical use to treat inflammation, we determined the effect of VAP-1 deletion on useful immune responses. Oral immunization with OVA showed defective T and B cell responses in VAP-1-deficient mice. Antimicrobial immune responses against Staphylococcus aureus and coxsackie B4 virus were also affected by the absence of VAP-1. Importantly, when the function of VAP-1 was acutely neutralized using small molecule enzyme inhibitors and anti-VAP-1 Abs rather than by gene deletion, no significant impairment in antimicrobial control was detected. In conclusion, VAP-1-deficient mice have mild deviations in the mucosal immune system and therapeutic targeting of VAP-1 does not appear to cause a generalized increase in the risk of infection.

Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury

Significance Organ fibrosis is a major cause of global morbidity and mortality. It is driven by chronic inflammation and associated oxidative stress with depletion of cellular antioxidant defenses. We demonstrate a pathway in which the evolutionarily conserved receptor stabilin-1 on tissue-infiltrating macrophages provides a second-line defense to prevent tissue damage from oxidative stress. Stabilin-1+ monocytes take up malondialdehyde-LDL (MDA-LDL), a major product of oxidative lipid peroxidation, to form ceroid-laden macrophages. Through the uptake of MDA-LDL, stabilin-1 suppresses production of the profibrogenic chemokine CCL3 and prevents excessive collagen deposition in experimental models of liver fibrosis. We propose that macrophage stabilin-1 is a critical defense against oxidative tissue damage and thereby maintains tissue homeostasis. Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1+ macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP+ fibrogenic cells. Stabilin-1−/− macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6Clo monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1+ monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage–specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1+ macrophages shape the tissue microenvironment during liver injury and healing.