The neuropeptide calcitonin gene-related peptide (CGRP) stimulates T cell migration into collagen matrices
Introduction
Lymphocytes can migrate to specific sites in tissues by passage of endothelial cells and subsequent infiltration of perivascular extracellular matrix (ECM) (Springer, 1994, Butcher and Picker, 1996). The capacity to migrate and localize in tissues is probably of vital importance for the protective function of lymphocytes against infectious agents. However, the capacity to migrate and infiltrate tissues is also a major reason why lymphocytes can cause autoimmunity, allergy and graft rejection or why neoplastic lymphocytes accumulate in tissues. It is important to identify the factors that induce T cell infiltration and accumulation in tissues during immunological disease processes and the inherent properties that render T cells infiltrative. T lymphocytes can migrate into/infiltrate three-dimensional (3D) matrices of collagen type I or matrigel and these substrata may be used as in vitro tissue models to analyze the mechanisms that render lymphocytes infiltrative (Wilkinson et al., 1984, Schor et al., 1983, Sundqvist et al., 1993, Ivanoff et al., 1999). The capacity to infiltrate 3D substrata is distinguishable from migration on two-dimensional (2D) substrates, such as plastic or through the holes of Boyden chamber filters (Ivanoff et al., 1999, Ivanoff et al., 2000). Thus, T cell infiltration seems to be a specific property distinguishable from migration per se as illustrated by the fact that virtually all leukemic T cell lines can migrate in Boyden assays but only some cell lines can penetrate a 3 D-matrix (Ivanoff et al., 2000). Infiltrative capacity correlates with the expression of matrix metalloproteinases (MMP's) indicating that degradation of ECM may be a prerequisite for the cells to penetrate (Ivanoff et al., 1999). However there is no remodeling of the matrix by the infiltrating lymphocytes indicating that MMP's do not function by the generation of holes for the penetrating lymphocytes (Wolf et al., 2003).
Lymphocyte migration/infiltration of tissue consists of several steps including adhesion to endothelial cells, transendothelial migration and migration to tissue sites (Baggiolini, 1998). The chemokine system has emerged as important regulators of all these steps (Hall, 1998, Sanchez-Madrid and del Pozo, 1999, Voermans et al., 2001). In humans, on the basis of expression of SDF-1α at sites of aberrant lymphocyte accumulation it has been suggested that SDF-1α is important in facilitating cell migration to inflamed tissues (Pablos et al., 1999, Buckley et al., 2000, Nanki et al., 2000). SDF-1α is a potent stimulator of T cell migration into three-dimensional type I collagen matrices as demonstrated using T cells fresh from the blood and T cell lines (Ivanoff et al., 2005).
Inflammatory responses are regulated by sensory neurons and stimulation of sensory neurons has been shown to reduce hypertension, stress-induced gastric mucosal injury, reperfusion-induced liver injury, and endotoxin-induced shock responses (Okajima and Harada, 2006). There is evidence that the nervous and the immune systems interact in the regulation of host defense and in pathological conditions and therefore it is important to further elucidate this neuroendocrine immune regulation (Wimalawansa, 1996, Poyner, 1993, Eskandari et al., 2003). Neuropeptides are a heterogenous group of several hundred biologically active peptides present in neurons of both the central and peripheral nervous system. Neuropeptides, such as calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), somatostatin, substance P and vasoactive intestinal peptide (VIP), play an important role in mediating communications between the nervous, endocrine and immune systems and neurotransmitters are released from nerve terminals at various extravasal sites to which T cells are attracted under inflammatory conditions (Sternberg, 2001). T cells express specific receptors for neurotransmitters on their surface (Reubi et al., 1998, Van Hagen, 1996) and all primary and secondary lymphoid organs are massively innervated (Stevens-Felten and Bellinger, 1997). Therefore, T cells are likely to be exposed to a variety of neurotransmitters. CGRP has previously been shown to exhibit chemotactic activity for T lymphocytes in a 2D microchemotaxis Boyden assay system (Foster et al., 1992). In this study we were particularly interested in neuropeptides as possible regulators of T cell migration since their role in this context is virtually unknown. CGRP was found to be a specific stimulator of T cell migration into 3D collagen type I matrices, whereas a number of other neuropeptides and opioids did not influence T cell infiltration.
Section snippets
Chemicals
α-CGRP, ß-CGRP, CGRP 8-37, somatostatin-14, NPY, substance P, VIP, β-endorphin and metenkephalin were purchased from Sigma Chemical Co., St. Louis, MO. SDF-1α, IL-2 and IL-4 were from R&D Systems Europé Ltd, Abington, UK. Mouse-anti human monoclonal antibodies against CD3 (clone SK 7, IgG1) and CD4 (clone RPA-T4) were from BD Biosciences, San Jose, CA. Mouse-anti human monoclonal antibodies against CD8 (clone C8/144B), CD45RA (clone 4KB5) and CD45RO (clone UCHL1) were from DakoCytomation,
CGRP stimulates migration/infiltration of T lymphocytes
To analyze the possible influence of neuropeptides and opioids on lymphocyte migration non-activated and anti-CD3 activated blood lymphocytes were allowed to migrate into type I collagen gels containing different neuropeptides or opioids (Fig. 1, Fig. 2). SDF-1α applied in the same way was used as a comparison. Lymphocyte migration/infiltration was determined by counting the number of cells at different depths of the collagen. CGRP present inside the collagen stimulated cell infiltration,
Discussion
This study presents new information regarding the crosstalk between the nervous and the immune system with special reference to the neuropeptide CGRP. CGRP appears to be a specific stimulator of T cell migration within a 3D collagen matrix whereas the other neuropeptides analyzed do not affect T cell migration. CGRP did not increase the migration capacity of B cells indicating that this neuropeptide selectively stimulates T cells. CGRP has receptors on T cells and T cells also produce and
Acknowledgements
This study was supported by grants from the Swedish Research Council (project 56X-20347), the Edvard Welander Foundation and the Finsen Foundation.
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