InVivoMAb anti-rat L-selectin (CD62L)

InVivoMAb anti-rat L-selectin (CD62L) (CLONE: OX-85)

Boieri M, Shah P, Jalapothu D, Zaitseva O, Walter L, Rolstad B, Naper C, Dressel R, Inngjerdingen M (2017). "Rat acute GvHD is Th1 driven and characterized by predominant donor CD4⁺ T-cell infiltration of skin and gut" Exp Hematol . PubMed

Acute graft-versus-host disease (aGvHD) remains a significant hurdle to successful treatment of many hematological disorders. The disease is caused by infiltration of alloactivated donor T cells primarily into the gastrointestinal tract and skin. Although cytotoxic T cells mediate direct cellular damage, T helper (Th) cells differentially secrete immunoregulatory cytokines. aGvHD is thought to be initiated primarily by Th1 cells but a consensus is still lacking regarding the role of Th2 and Th17 cells. The aim of this study was to determine the contribution of distinct T-cell subsets to aGvHD in the rat. aGvHD was induced by transplanting irradiated rats with T-cell-depleted major histocompatibility complex-mismatched bone marrow, followed 2 weeks later by donor lymphocyte infusion. Near complete donor T-cell chimerism was achieved in the blood and lymphatic tissues, in contrast to mixed chimerism in the skin and gut. Skin and gut donor T cells were predominantly CD4⁺, in contrast to T cells in the blood and lymphatic tissues. Genes associated with Th1 cells were upregulated in gut, liver, lung, and skin tissues affected by aGvHD. Increased serum levels of CXCL10 and IL-18 preceded symptoms of aGvHD, accompanied by increased responsiveness to CXCL10 by blood CD4⁺ T cells. No changes in the expression of Th2- or Th17-associated genes were observed, indicating that aGvHD in this rat model is mainly Th1 driven. The rat model of aGvHD could be instrumental for further investigations of donor T-cell subsets in the skin and gut and for exploring therapeutic options to ameliorate symptoms of aGvHD.

Kühl AA, Kakirman H, Janotta M, Dreher S, Cremer P, Pawlowski NN, Loddenkemper C, Heimesaat MM, Grollich K, Zeitz M, Farkas S, Hoffmann JC (2007). "Aggravation of different types of experimental colitis by depletion or adhesion blockade of neutrophils" Gastroenterology 133(6):1882-92. PubMed

Background & aims: Neutrophils are generally thought to play an important proinflammatory role in the pathogenesis of inflammatory bowel disease. The objective of this study was to evaluate whether blocking the invasion of neutrophils by anti-L-selectin monoclonal antibodies modulates chemically induced colitis and how this modulation is accomplished. Methods: Trinitrobenzene sulfonic acid/dinitrobenzene sulfonic acid (TNBS/DNBS)-induced colitis was studied in rats on treatment with anti-L-selectin monoclonal antibodies (mAb) or antineutrophil antiserum. Different anti-L-selectin mAb, either blocking or nonblocking, as well as F(ab)(2) fragments were evaluated. Additionally, leukocyte migration was examined using intravital microscopy. Furthermore, the effect of neutrophil depletion in rat TNBS-induced colitis was studied either prior to or after colitis induction as well as murine CD4(+)CD45RB(high) transfer colitis. Finally, bacterial translocation during DNBS-induced colitis was studied in neutrophil-depleted and control rats. Results: Anti-L-selectin mAb treatment resulted in increased mortality and bowel inflammation as well as hemorrhagic eye secretion. No clear difference was found between blocking and nonblocking mAb or F(ab)(2) fragments. For all investigated antibodies/fragments, either complete blockade of leukocyte invasion or marked neutrophil depletion was found. Accordingly, neutrophil depletion by antiserum resulted in aggravation of rat DNBS-induced colitis as well as murine transfer colitis. Conclusions: Adhesion blockade or neutrophil depletion aggravates rat TNBS/DNBS-induced colitis together with extraintestinal manifestations of the eyes. Therefore, neutrophils appear to have an important role in mucosal repair processes. Importantly, adhesion blockade as a therapeutic concept can be detrimental in inflammatory bowel disease.

Milićević NM, Nohroudi K, Milićević Z, Westermann J (2004). "Blood lymphocytes, monocytes and NK cells modulate their expression of CD44, ICAM-1, LFA-1 and MHC class II after arrival into lymphoid organs" Immunol Invest 33(4):439-52. PubMed

Adhesion molecules expressed on surface membranes of lymphocytes and other leukocytes enable their entry into the lymphoid and other tissues. However, little is known about molecules that govern the transit of leukocytes through the parenchyma of lymphoid organs proper. We show that in comparison to blood leukocytes, the corresponding cells isolated from lymphoid organs, i.e., lymph nodes and spleen, have a significantly augmented expression of certain surface molecules. The helper and cytotoxic subsets of T cells, as well as B cells, display the increased expression of CD44, ICAM-1 and LFA-1, whereas B cells additionally show the augmented expression of MHC class II. In comparison with blood monocytes, splenic monocytes show the increased expression of ICAM-1 and MHC class II molecules. When compared with blood NK cells, splenic NK cells only show the increased expression of ICAM-1. The molecules, which we show to be up regulated upon the entry of leukocytes into lymphoid organs, could be involved in their retention within the tissue via cell-cell or cell-extracellular matrix interactions and in control of their transit through lymphoid tissues.

Milićević NM, Nohroudi K, Milićević Z, Westermann J (2004). "Blood lymphocytes, monocytes and NK cells modulate their expression of CD44, ICAM-1, LFA-1 and MHC class II after arrival into lymphoid organs" Immunol Invest 33(4):439-52. PubMed

Adhesion molecules expressed on surface membranes of lymphocytes and other leukocytes enable their entry into the lymphoid and other tissues. However, little is known about molecules that govern the transit of leukocytes through the parenchyma of lymphoid organs proper. We show that in comparison to blood leukocytes, the corresponding cells isolated from lymphoid organs, i.e., lymph nodes and spleen, have a significantly augmented expression of certain surface molecules. The helper and cytotoxic subsets of T cells, as well as B cells, display the increased expression of CD44, ICAM-1 and LFA-1, whereas B cells additionally show the augmented expression of MHC class II. In comparison with blood monocytes, splenic monocytes show the increased expression of ICAM-1 and MHC class II molecules. When compared with blood NK cells, splenic NK cells only show the increased expression of ICAM-1. The molecules, which we show to be up regulated upon the entry of leukocytes into lymphoid organs, could be involved in their retention within the tissue via cell-cell or cell-extracellular matrix interactions and in control of their transit through lymphoid tissues.

Mannie MD, Dawkins JG, Walker MR, Clayson BA, Patel DM (2004). "MHC class II biosynthesis by activated rat CD4+ T cells: development of repression in vitro and modulation by APC-derived signals" Cell Immunol 230(1):33-43. PubMed

This study focused on synthesis of MHC class II glycoproteins (MHCII) by rat CD4(+) T-helper cells. During activation in Con A and IL-2, purified rat splenic CD4(+) T cells expressed abundant surface MHCII together with transcripts for I-A alpha/beta, invariant chain, and the type III and type IV MHC class II transactivator (CIITA). Activated thymic CD8(+)CD4(-) and CD8(+)CD4(+) T cells exhibited essentially the same phenotype. MHCII synthesis by CD4(+) T cells enabled presentation of myelin basic protein (MBP) to antigen-specific responders. T cell expression of MHCII was due to direct biosynthesis rather than adsorption from professional APC; indeed, T cell-mediated expression of MHCII was optimal in the absence of professional APC. Despite periodic reactivation with Con A during 3-4 weeks of culture, CD4(+) T cells repressed MHCII synthesis and reverted to a MHCII(-) phenotype. These short-term lines resembled established lines of MBP-specific T cells in that mitogenic activation elicited extensive blastogenesis without MHCII synthesis. Activation-dependent synthesis of MHCII however was partially restored in lines of mitogen-stimulated T cells when the cultures were reconstituted with irradiated splenic APC. These data indicate that most naive rat CD4(+) T cells exhibit activation-dependent synthesis of MHCII whereas continuously propagated T cells require an APC-derived signal to support MHCII synthesis.

Teeling JL, Bleeker WK, Rigter GM, van Rooijen N, Kuijpers TW, Hack CE (2001). "Intravenous immunoglobulin preparations induce mild activation of neutrophils in vivo via triggering of macrophages--studies in a rat model" Br J Haematol 112(4):1031-40. PubMed

Despite widespread use in various immune disorders, the in vivo mechanisms of action of intravenous immunoglobulin (IVIG) preparations are not well known. We previously reported that human neutrophils degranulate after incubation with IVIG in vitro as a result of interaction with FcgammaRII. The purpose of this study was to determine whether IVIG might stimulate neutrophils in vivo. Anaesthetized rats received a bolus intravenous injection of IVIG preparations, containing either high (aged IVIG) or low (fresh IVIG) amounts or IgG dimers at a dose of 250 mg/kg. Administration of aged IVIG induced neutrophil activation in vivo, whereas no effect was observed after infusion of fresh IVIG. Histological examination of lung tissue demonstrated mild influx of neutrophils into the pulmonary tissue after aged IVIG administration, though gross damage did not occur. Macrophage-depleted rats no longer showed activation of neutrophils after infusion of aged IVIG, suggesting that neutrophils become activated via an indirect macrophage dependent way. We conclude that IVIG induces a mild activation of neutrophils in vivo via triggering of macrophages depending on the amount of IgG dimers. For this reason, IVIG preparations with a high content of dimers may not always be as harmless as generally believed and may be responsible for some of the side-effects observed during IVIG infusions.

Milićević NM, Luettig B, Trautwein C, Wüstefeld T, Mähler M, Jecker P, Wonigeit K, Westermann J (2001). "Splenectomy of rats selectively reduces lymphocyte function-associated antigen 1 and intercellular adhesion molecule 1 expression on B-cell subsets in blood and lymph nodes" Blood 98(10):3035-41. PubMed

Splenectomy increases the number of B cells in the blood of humans and animals. It is unknown whether this is due to changes in migration, proliferation, or both. The numbers of naïve (IgD(+)IgM(+)), memory (IgD(-)IgM(high)), newly formed (IgM(high)CD90(high)), early recirculating follicular (IgM(low)CD90(high)), recirculating follicular (IgM(low)CD90(-)), and marginal zone (IgM(high)CD90(-)) phenotype B cells were determined in control and splenectomized rats by flow cytometry. All subsets increased significantly in the blood after splenectomy. Because surface molecules are involved in the regulation of migration and proliferation, their expression (lymphocyte function-associated antigen 1 [LFA-1], intercellular adhesion molecule 1 (ICAM-1), L-selectin, alpha4-integrins, CD44, major histocompatability complex class II, interleukin 2 receptor-alpha chain) was determined on B- and T-cell subsets of both groups. B cells, but not T cells, showed a significantly reduced LFA-1 and ICAM-1 expression in blood and lymph nodes, whereas the expression of the other surface molecules analyzed remained unchanged. The down-regulation of these molecules did not influence the adherence of B cells to high endothelial venules in vitro. In vivo, however, ICAM-1(low)-expressing B cells migrated significantly faster through lymph nodes (ICAM-1(low) 41 +/- 5 hours versus ICAM-1(high) 58 +/- 3 hours), whereas proliferation of B cells in bone marrow, lymph node, and blood remained unchanged. Thus, the presence of one organ is necessary for appropriate expression of LFA-1 and ICAM-1 on B cells in other, distant organs. The more rapid transit of ICAM-1(low) B cells through lymph nodes may be responsible for the increased B-cell number in the blood after splenectomy.

Sanderson P, Thies F, Calder PC (2000). "Extracellular release of free fatty acids by rat T lymphocytes is stimulus-dependent and is affected by dietary lipid manipulation" Cell Biochem Funct 18(1):47-58. PubMed

[(3)H]-Arachidonic acid-labelled rat T lymphocytes released radioactivity extracellularly when stimulated by the calcium ionophore A23187 or by monoclonal antibodies to some cell surface structures (CD2, CD5, CD11a, CD18, CD54, T-cell receptor) but not to others (CD49d, CD62L); release was greater with the calcium ionophore. Almost all of the radioactivity released from anti-CD2-stimulated lymphocytes was recovered in the free fatty acid fraction, whereas only about 50 per cent of that released after A23187 stimulation was recovered in this fraction. A23187 stimulation resulted in release of arachidonic acid from a variety of phospholipids (phosphatidylinositol, phosphatidylcholine and perhaps phosphatidylethanolamine), while the monoclonal antibody stimulation released arachidonic acid from phosphatidylinositol and perhaps phosphatidylcholine. Unstimulated lymphocytes released a range of fatty acids extracellularly, with palmitic acid accounting for 35-40 per cent and arachidonic acid for 5 per cent of released fatty acid. Stimulation of lymphocytes with either anti-CD2 or A23187 increased total fatty acid release 1.5- to 1.8-fold. In both cases palmitic acid remained the most predominant fatty acid released but the contribution of arachidonic acid increased. The type of lipid fed to the rats significantly influenced the amount and type of fatty acid released. Fish oil feeding significantly reduced extracellular fatty acid release by stimulated lymphocytes.

Nicholson MW, Barclay AN, Singer MS, Rosen SD, van der Merwe PA (1998). "Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1" J Biol Chem 273(2):763-70. PubMed

The selectin family of cell adhesion molecules mediates the tethering and rolling of leukocytes on blood vessel endothelium. It has been postulated that the molecular basis of this highly dynamic adhesion is the low affinity and rapid kinetics of selectin interactions. However, affinity and kinetic analyses of monomeric selectins binding their natural ligands have not previously been reported. Leukocyte selectin (L-selectin, CD62L) binds preferentially to O-linked carbohydrates present on a small number of mucin-like glycoproteins, such as glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), expressed in high endothelial venules. GlyCAM-1 is a soluble secreted protein which, following binding to CD62L, stimulates beta2-integrin-mediated adhesion of lymphocytes. Using surface plasmon resonance, we show that a soluble monomeric form of CD62L binds to purified immobilized GlyCAM-1 with a dissociation constant (Kd) of 108 microM. CD62L dissociates from GlyCAM-1 with a very fast dissociation rate constant (>/=10 s-1) which agrees well with the reported dissociation rate constant of CD62L-mediated leukocyte tethers. The calculated association rate constant is >/=10(5) M-1 s-1. At concentrations just above its mean serum level (approximately 1.5 microg/ml or approximately 30 nM), GlyCAM-1 binds multivalently to immobilized CD62L. It follows that soluble GlyCAM-1 may cross-link CD62L when it binds to cells, suggesting a mechanism for signal transduction.