Karl-Gösta Sundqvist

Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages

ABSTRACT Cytolysin A (ClyA) is a newly discovered cytolytic protein ofEscherichia coli K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.

Abnormal expression of chemokine receptors on T-cells from patients with systemic lupus erythematosus

The expressionof chemokinereceptors on T-cells and chemokinelevels in the blood was studied in 23 patients with SLE (ACR criteria), seven patients with rheumatoid arthritis (RA) and in 15 healthy controls using flow cytometry, RT-PCR and ELISA. The cell surface expression of the chemokine receptors CXCR5 and CCR6 was decreased in SLE patients compared with controls (P 0.051 and P 0.002, respectively). The decrease of CXCR5 was confined to SLE patients with inactive disease (SLEDAI < 6) compared with active disease (SLEDAI ≥6) and controls. CXCR2 and CCR1 were increased in patients with active SLE compared with patients with inactive disease (P 0.001 and P 0.01, respectively) and with controls (P 0.02 and P 0.053, respectively). The levels of the chemokines MIP-1b MCP-1, SDF-1a, IP-10 and RANTES were significantly elevated in SLE patients compared with controls. Patients with renal involvement had increased surface expression of CXCR3 and CCR3 (P 0.04 in both) and a lower level of soluble IP-10 compared with patients without renal disease (P 0.025) and compared with controls (P 0.001). The ratio between CCR5 and CCR3 was significantly increased in RA patients compared with SLE patients and controls supporting a Th1 overweight in RA. In conclusion, patients with SLE showed abnormal T-cell expression of several chemokine receptors and levels of soluble chemokines in their plasma/serum.

A CD26-Controlled Cell Surface Cascade for Regulation of T Cell Motility and Chemokine Signals

Chemokines are key regulators of cell trafficking, and dipeptidyl peptidase IV/CD26 (CD26) inactivates chemokines. Here we show that the CD26-processed chemokines SDF1α/CXCL12 and RANTES/CCL5, in contrast to a control chemokine not processed by CD26, are potent inducers of cell surface expression of thrombospondin-1 (TSP-1) in T lymphocytes through a CD26-controlled mechanism and that TSP-1 stimulates expression of lipoprotein receptor related protein/CD91. Accordingly, intact TSP-1 and a peptide mimetic of a sequence in TSP-1 were sufficient to stimulate CD91 expression. The chemokine-induced expression of TSP-1 and CD91 was mimicked by inhibitors of CD26 and CXCL12 and CCL5 as well as inhibitors of CD26 stimulated polarized cytoplasmic spreading and migration through TSP-1. Silencing of CD26 using small interfering RNA or Ab-induced modulation of CD26 also increased TSP-1 expression and enhanced cytoplasmic spreading and T cell migration markedly. These results indicate that CD26 is an endogenous inhibitor of T cell motility through inhibition of TSP-1 expression and that chemokines stimulate cell polarity and migration through abrogation of the CD26-dependent inhibition. This suggests that T cell motility is regulated by a cascade of interacting cell surface molecules.

Infiltrative capacity of T leukemia cell lines: a distinct functional property coupled to expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1).

Infiltrative capacity was found to distinguish separate T leukemia cell lines. Of seven T-cell lines four exhibited capacity to infiltrate Matrigel. Analysis of infiltration was performed at the single-cell level throughout the Matrigel using a depth meter. Further, we examined differences in migration capacity and metalloproteinase production between infiltrating and non-infiltrating T-cell lines. The capacity to infiltrate was not directly correlated to the capacity to adhere to the Matrigel or to migrate on/to extracellular matrix components. It is concluded that infiltration capacity does not simply reflect capacity to migrate but represents a distinct functional property. The production of metalloproteinases and their inhibitors by the separate T-cell lines was analyzed using rt PCR, biosynthetic labelling, zymography, immunoprecipitation and ELISA. All T-cell lines with capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) while non-infiltrating cell lines did not express MMP-9. Expression of MMP-1, 2, 3, 10, 14 and 17 showed no correlation to capacity to infiltrate. Analysis of infiltration in the presence of a metalloprotease inhibitor showed an increased number of cells within the gel. This enhancement of infiltration suggests that the function of MMPs and/or their inhibitors in lymphocyte infiltration is more complex than previously thought.

The neuropeptide calcitonin gene-related peptide (CGRP) stimulates T cell migration into collagen matrices

CGRP significantly stimulated migration of non-activated and anti-CD3 activated T lymphocytes into a collagen matrix when present inside the collagen, whereas somatostatin-14, NPY, substance P, VIP, beta-endorphin and metenkephalin had no or little effect. The CGRP antagonist CGRP 8-37 abrogated the CGRP-induced cell infiltration. Virtually all migrating cells were CD3+ (>96%) and CGRP did not stimulate B-cell migration. The migration capacity showed no selective relationship to the expression of CD4+, CD8+, CD45RO+ (memory), or CD45RA+ (naive) T cell markers indicating that the regulation of T cell migration is distinct from that of the major T cell phenotypes.

The role of chemokines and extracellular matrix components in the migration of T lymphocytes into three-dimensional substrata

The role of chemokines and their interactions with extracellular matrix components (ECM) or the capacity of T cells to migrate into and accumulate within three‐dimensional (3D) collagen type 1 substrata was studied. We examined the influence of chemokines and fibronectin on the infiltration properties of non‐infiltrative (do not migrate into 3D substrata) and spontaneously infiltrative (migrate into 3D substrata) T‐cell lines. Infiltrative and non‐infiltrative T‐acute lymphocytic leukaemic cell lines exhibited no consistent differences with respect to the expression of various chemokine receptors or β1‐integrins. Chemokines presented inside the collagen increased the depth of migration of infiltrative T‐cell lines, but did not render non‐infiltrative T‐cell lines infiltrative, although they augmented the attachment of non‐infiltrative T‐cell lines to the upper surface of the collagen. The presence of fibronectin inside the collagen did not render non‐infiltrative T‐cell lines infiltrative, but markedly augmented the migration of ‘infiltrative’ T‐cell lines into collagen. Both infiltrative and non‐infiltrative T‐cell lines showed migratory responses to chemokines in Boyden assays (migration detected on 2D substrata). These results indicate that the process of T‐cell infiltration/migration into 3D substrata depends on a tissue penetration mechanism distinguishable from migration on 2D substrata and that the basic capacity of T cells to infiltrate is independent of chemokines and ECM components applied as attractants.

Changes in chemokines and their receptors in blood during treatment with the TNF inhibitor infliximab in patients with rheumatoid arthritis

Objectives: Chemokines are involved in leucocyte recruitment into inflammatory sites. The release of certain chemokines is augmented by tumour necrosis factor (TNF). Infliximab, a monoclonal antibody that blocks the effects of TNF, is used for treatment of rheumatoid arthritis (RA). The effect of TNF blockage on chemokines is not fully understood. The aim of this study was to analyse the effects on chemokines and their receptors on peripheral mononuclear cells of anti-TNF treatment in RA patients. Method: Twelve patients with established RA who started treatment with infliximab and nine patients with early RA treated with other anti-rheumatic drugs were followed clinically for 30 weeks and chemokine levels in blood samples were analysed along with chemokine receptor expression on the surface of T cells and monocytes. Nine healthy subjects were included as a control group. Results: The chemokine CXCL10/IP-10 was significantly higher in RA patients than in healthy controls (p = 0.012). Two weeks after infliximab infusion, CXCL10/IP-10, CCL2/MCP-1, and CCL4/MIP-1β had decreased significantly (p = 0.005, 0.037, and 0.028, respectively), and after 30 weeks of treatment, soluble CD26 was significantly increased (p = 0.050). Several chemokine receptors on T cells were elevated in RA patients at inclusion. The expression of CCR2 and CXCR1 on T cells decreased significantly after infliximab treatment. Conclusions: The chemokines CXCL10/IP-10, CCL2/MCP-1, and CCL4/MIP-1β, mainly targeting the T-helper (Th)1 immune response, decreased after treatment with anti-TNF, suggesting a more pronounced effect on Th1 activity than on Th2-mediated response. Several chemokine receptors on blood T cells were elevated in RA patients, suggesting that they may be involved in the recruitment of T lymphocytes from the blood to affected tissues.

Excess glucose induces hypoxia-inducible factor-1α in pancreatic cancer cells and stimulates glucose metabolism and cell migration.

Pancreatic cancer patients frequently show hyperglycemia, but it is uncertain whether hyperglycemia stimulates pancreatic cancer cells. We have investigated whether excess glucose induces hypoxia-inducible factor-1α (HIF-1α) and stimulates glucose metabolism and cell migration in pancreatic cancer cells. We studied wild-type (wt) MiaPaCa2 pancreatic cancer cells and a MiaPaCa2 subline (namely si-MiaPaCa2) that had HIF-1α-specific small interfering RNA. Wt-MiaPaCa2 cells are known to be HIF-1α-positive in hypoxia and HIF-1α-negative in normoxia, whereas si-MiaPaCa2 cells are devoid of HIF-1α in both normoxia and hypoxia. We incubated these cells with different amounts of glucose and determined HIF-1α mRNA and protein by real-time polymerase chain reaction and western blotting. We determined glucose consumption, lactate production and intracellular hexokinase-II and ATP to assess glucose metabolisms and determined pyruvate dehydrogenase kinase-1, reactive oxygen species and fumarate to assess mitochondrial activities. Further, we studied cell migration using a Boyden chamber. Excess glucose (16.7−22.2mM) increased HIF-1α in hypoxic wt-MiaPaCa2 cells. HIF-1α expression increased ATP contents and inhibited mitochondrial activities. Extracellular glucose and hypoxia stimulated glucose metabolisms independent of HIF-1α. Excess glucose stimulated the migration of wt- and si-MiaPaCa2 cells in both normoxia and hypoxia. Thus, glucose stimulated cell migration independent of HIF-1α. Nevertheless, hypoxic wt-MiaPaCa2 cells showed greater migrating ability than their si-MiaPaCa2 counterparts. We conclude that (1) excess glucose increases HIF-1α and ATP in hypoxic wt-MiaPaCa2 cells, (2) extracellular glucose and hypoxia regulate glucose metabolisms independent of HIF-1α and (3) glucose stimulates cell migration by mechanisms that are both dependent on HIF-1α and independent of it.

T-cell expression of CD91 - a marker of unresponsiveness to anti-TNF therapy in rheumatoid arthritis.

Eriksson C, Rantapää‐Dahlqvist S, Sundqvist K‐G. T‐cell expression of CD91 – a marker of unresponsiveness to anti‐TNF therapy in rheumatoid arthritis. APMIS 2010; 118: 837–45.

Receptor communication within the lymphocyte plasma membrane: a role for the thrombospondin family of matricellular proteins.

Abstract.Lymphocytes, the principal cells of the immune system, carry out immune surveillance throughout the body by their unique capacity to constantly reposition themselves between a free-floating vascular state and a tissue state characterized by migration and frequent adhesive interactions with endothelial cells and components of the extracellular matrix. Therefore, mechanisms co-ordinating adhesion and migration with signals delivered through antigen recognition probably play a pivotal role for the regulation of lymphocyte behaviour and function. Endogenous thrombospondin-1 (TSP-1) seems to be the hub in such a mechanism for autocrine regulation of T cell adhesion and migration. TSP-1 functions as a mediator of cis interaction of vital receptors within the T lymphocyte plasma membrane, including integrins, low density lipoprotein receptor-related protein, calreticulin and integrin-associated protein.