Thomas B. Issekutz

Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions

The tissues of the central nervous system are effectively shielded from the blood circulation by specialized vessels that are impermeable not only to cells, but also to most macromolecules circulating in the blood. Despite this seemingly absolute seclusion, central nervous system tissues are subject to immune surveillance and are vulnerable to autoimmune attacks. Using intravital two-photon imaging in a Lewis rat model of experimental autoimmune encephalomyelitis, here we present in real-time the interactive processes between effector T cells and cerebral structures from their first arrival to manifest autoimmune disease. We observed that incoming effector T cells successively scanned three planes. The T cells got arrested to leptomeningeal vessels and immediately monitored the luminal surface, crawling preferentially against the blood flow. After diapedesis, the cells continued their scan on the abluminal vascular surface and the underlying leptomeningeal (pial) membrane. There, the T cells encountered phagocytes that effectively present antigens, foreign as well as myelin proteins. These contacts stimulated the effector T cells to produce pro-inflammatory mediators, and provided a trigger to tissue invasion and the formation of inflammatory infiltrations.

Chronic inflammation upregulates chemokine receptors and induces neutrophil migration to monocyte chemoattractant protein-1

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that stimulates monocyte recruitment when injected into tissues of healthy animals. However, the function of this chemokine in models with preexisting inflammation is not known. Therefore, MCP-1 was superfused over the mesentery of naive rats or rats with chronic adjuvant-induced vasculitis. MCP-1 elicited increased leukocyte transendothelial migration in adjuvant-immunized rats compared with naive animals. Surprisingly, histology revealed that neutrophils constituted the majority of leukocytes recruited in adjuvant-immunized animals. In vitro, MCP-1 was also able to induce chemotaxis of neutrophils isolated from adjuvant-immunized rats but not from naive rats. Flow cytometry revealed novel expression of the CC chemokine receptors CCR1 and CCR2 on neutrophils from adjuvant-immunized animals. In naive animals, an antibody against CD18 blocked leukocyte adhesion and emigration in response to MCP-1. In adjuvant-immunized animals, leukocyte adhesion was reduced by antibodies against the alpha4-integrin but not by antibodies against CD18. However, the CD18 antibody did block emigration. To our knowledge, this study is the first to show increased sensitivity to a CC chemokine in a model with preexisting inflammation, and altered leukocyte recruitment profiles in response to MCP-1. It also demonstrates that CD18 is required for chemokine-induced leukocyte transendothelial migration, independent of its known role in mediating firm adhesion. J. Clin. Invest. 103:1269-1276 (1999).

Requirements for leukocyte adhesion molecules in nephrotoxic nephritis.

Requirements for leukocyte adhesion molecules as well as cytokines have been determined in the rat model of acute nephrotoxic nephritis. Proteinuria (at 24 h) and neutrophil accumulation in renal glomeruli (at 6 h) have been used as the endpoints. For full accumulation in glomeruli of neutrophils as well as full development of proteinuria, requirements have been demonstrated for TNF alpha, (but not IL-1), CD11b (but not CD11a), very late arising-4 (CD49d/CD29), and intercellular adhesion molecule-1 but not endothelial leukocyte adhesion molecule-1 (E-selectin). By immunohistochemical approaches, infusion of antibody to glomerular basement membrane induced glomerular upregulation of intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular adhesion molecule-1. Treatment of rats with anti-TNF alpha or soluble recombinant human TNF receptor-1 blocked this expression. Renal arterial infusion of TNF alpha induced glomerular expression of all three endothelial adhesion molecules, but infusion of IL-1 beta did not. These data suggest that, in neutrophil and complement-dependent anti-glomerular basement membrane-induced acute nephritis in rats, there are selective requirements for cytokines, beta 1 and beta 2 integrins, and endothelial adhesion molecules. These requirements contrast with those found in other vascular beds in which complement and neutrophil-induced vascular injury has been induced by deposition of immune complexes.

Human mast cell activation with virus-associated stimuli leads to the selective chemotaxis of natural killer cells by a CXCL8-dependent mechanism

Human mast cells are found in skin and mucosal surfaces and next to blood vessels. They play a sentinel cell role in immunity, recognizing invading pathogens and producing proinflammatory mediators. Mast cells can recruit granulocytes, and monocytes in allergic disease and bacterial infection, but their ability to recruit antiviral effector cells such as natural killer (NK) cells and T cells has not been fully elucidated. To investigate the role of human mast cells in response to virus-associated stimuli, human cord blood-derived mast cells (CBMCs) were stimulated with polyinosinic.polycytidylic acid, a double-stranded RNA analog, or infected with the double-stranded RNA virus, reovirus serotype 3 Dearing for 24 hours. CBMCs responded to stimulation with polyinosinic.polycytidylic acid by producing a distinct chemokine profile, including CCL4, CXCL8, and CXCL10. CBMCs produced significant amounts of CXCL8 in response to low levels of reovirus infection, while both skin- and lung-derived fibroblasts were unresponsive unless higher doses of reovirus were used. Supernatants from CBMCs infected with reovirus induced substantial NK cell chemotaxis that was highly dependent on CXCL8 and CXCR1. These results suggest a novel role for mast cells in the recruitment of human NK cells to sites of early viral infection via CXCL8.

Human Mast Cells Transmigrate Through Human Umbilical Vein Endothelial Monolayers and Selectively Produce IL-8 in Response to Stromal Cell-Derived Factor-1α

Mature mast cells are generally considered to be less mobile cells residing within tissue sites. However, mast cell numbers are known to increase in the context of inflammation, and mast cells are recognized to be important in regulating local neutrophil infiltration. CXC chemokines may play a critical role in this process. In this study two human mast cell-like lines, HMC-1 and KU812, and human cord blood-derived primary cultured mast cells were employed to examine role of stromal cell-derived factor-1 (SDF-1) in regulating mast cell migration and mediator production. It was demonstrated that human mast cells constitutively express mRNA and protein for CXCR4. Stimulation of human mast cells with SDF-1, the only known ligand for CXCR4, induced a significant increase in intracellular calcium levels. In vitro, SDF-1α mediated dose-dependent migration of human cord blood-derived mast cells and HMC-1 cells across HUVEC monolayers. Although SDF-1α did not induce mast cell degranulation, it selectively stimulated production of the neutrophil chemoattractant IL-8 without affecting TNF-α, IL-1β, IL-6, GM-CSF, IFN-γ, or RANTES production, providing further evidence of the selective modulation of mast cell function by this chemokine. These findings provide a novel, SDF-1-dependent mechanism for mast cell transendothelial migration and functional regulation, which may have important implications for the local regulation of mast cells in disease.

The α4β1 (Very Late Antigen (VLA)-4, CD49d/CD29) and α5β1 (VLA-5, CD49e/CD29) Integrins Mediate β2 (CD11/CD18) Integrin-Independent Neutrophil Recruitment to Endotoxin-Induced Lung Inflammation

The β2 integrin cell adhesion molecules (CAM) mediate polymorphonuclear leukocyte (PMNL) emigration in most inflamed tissues, but, in the lung, other yet to be identified CAMs appear to be involved. In Lewis rats, the intratracheal injection of Escherichia coli-LPS induced acute (6-h) PMNL accumulation in the lung parenchyma (280 × 106 by myeloperoxidase assay; PBS control = 35 × 106) and bronchoalveolar lavage fluid (BALF = 27 × 106; PBS = 0.1 × 106). Parenchymal accumulation was not inhibited by a blocking Ab to β2 integrins and only minimally inhibited (20.5%; p < 0.05) in BALF. We examined the role of α4β1 and α5β1 integrins and of selectins in this PMNL recruitment. Treatment with mAbs to α4β1 or α5β1, even in combination, had no effect on PMNL accumulation induced by intratracheal LPS. However, anti-α4 combined with anti-β2 mAbs inhibited PMNL recruitment to the parenchyma by 56% (p < 0.001) and to BALF by 58% (p < 0.01). The addition of anti-α5 mAb to β2 plus α4 blockade inhibited PMNL accumulation further (by 79%; p < 0.05). In contrast, blockade of L-, P-, and E-selectins in combination or together with β2, α4, and α5 integrins had no effect. LPS-induced BALF protein accumulation was not inhibited by treatment with anti-β2 plus α4 mAbs, but was prevented when α5β1 was also blocked. Thus, while selectins appear to play no role, α4β1 and α5β1 function as major alternate CAMs to the β2 integrins in mediating PMNL migration to lung and to pulmonary vascular and epithelial permeability.

Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late activation antigen-4 integrins suppresses neutrophil and T-lymphocyte migration to the joints and improves clinical disease.

The migration of leucocytes from blood into the joint is a key feature of human and experimental arthritis. Adhesion molecules on leucocytes and vascular endothelium are important in this process and may be therapeutic targets for intervention in arthritis. We investigated whether monoclonal antibody treatment to block the α4 integrin, very late activation antigen‐4 (VLA‐4), and β2 integrins (CD11/CD18) administered to rats during the preclinical (day 5) or clinical phase (day 10+) would modify disease. When treatment was initiated 5 days after induction of disease, development of arthritis was significantly reduced by either anti‐α4 (TA‐2) or anti‐β2 (WT.3) monoclonal antibodies (mAb) and the combination of both mAb was even more effective (clinical scores: control 11.4; anti‐α4 6.6; anti‐β2 6.8; anti‐α4 + anti‐β2 3.9). When treatment was delayed until arthritis was apparent (day 10), the anti‐α4 + anti‐β2 mAb combination still significantly diminished the arthritis score on day 14 (control 13; anti‐α4 + anti‐β2 7.9). Treatment with anti‐α4 + anti‐β2 mAb decreased the migration to the joints of blood polymorphonuclear leucocytes (PMNL) by 66–79% and of spleen T lymphocytes by 56–75%, depending on the joint. In contrast, PMNL migration was abolished (>98%) and T‐cell migration markedly (87%) inhibited to dermal inflammatory reactions in the same animals. These findings demonstrate: that blocking mAb to α4 and β2 integrins can reduce the severity of adjuvant arthritis, even after joint inflammation has developed; that this treatment can markedly inhibit PMNL and T‐lymphocyte migration to the joints; and that yet to be defined mechanisms distinct from α4 (CD49d) and β2 (CD11/CD18) integrins, also contribute to leucocyte migration to inflamed joints. Identifying these additional adhesion mechanisms may be required to control joint inflammation further.

T lymphocyte migration to arthritic joints and dermal inflammation in the rat: Differing migration patterns and the involvement of VLA-4

The migration of T lymphocytes into arthritic joints of rats with adjuvant arthritis was examined and compared to the accumulation of the same cells in cutaneous inflammation, since previous studies had shown that only a subpopulation of T lymphocytes, found enriched in peritoneal exudates (sPEL), migrated efficiently to cutaneous inflammatory sites. Surprisingly, lymphocyte migration to the inflamed joint included T cells from most of the recirculating lymphocyte pool, including sPEL, spleen, peripheral lymph node (PLN), and Peyers patches, and was much more rapid than migration through either cutaneous sites or PLNs. Treatment of sPEL with antibody to VLA-4 inhibited sPEL accumulation in the joints, while anti-VLA-4 treatment did not affect the accumulation of PLN T cells. It is concluded that the arthritic joint not only attracts inflammation-seeking lymphocytes (sPEL), through at least a partially VLA-4 dependent interaction, but also large numbers of lymphocytes which normally migrate to PLNs through a VLA-4-independent mechanism.

Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

Blockade of Chemokine Receptor CXCR3 Inhibits T Cell Recruitment to Inflamed Joints and Decreases the Severity of Adjuvant Arthritis

T lymphocytes expressing the chemokine receptors, CCR2, CCR5, CXCR3, and CXCR6 are increased in inflamed tissues in rheumatoid arthritis. The role of CXCR3 in autoimmune arthritis induced in Lewis rats was investigated. CXCR3+ T cells migrated 2- to 3-fold more than CXCR3− T cells to inflamed joints in arthritic animals. CXCR3-expressing in vivo Ag-activated T lymphoblasts and in vitro-activated lymph node cells from arthritic animals were strongly recruited to the arthritic joints, and treatment with anti-CXCR3 mAb significantly inhibited this T cell recruitment by 40–60%. Immune T cells from the spleen and lymph nodes of actively immunized arthritic donors adoptively transferred arthritis to naive rats. Treatment with anti-CXCR3 mAb delayed the onset of arthritis and significantly reduced the severity of joint inflammation with a >50% decrease in the clinical arthritis score. Blockade of CXCR3 also significantly reduced the weight loss in the arthritic animals and inhibited neutrophil accumulation in the joints by 50–60%. There was a marked reduction in the leukocyte infiltration of the synovium in the presence of CXCR3 blockade and a decrease in the loss of articular cartilage of the joints. In conclusion, CXCR3 on T cells has an essential role in T cell recruitment to inflamed joints and the development of joint inflammation in adjuvant arthritis.