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.
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.
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 α 4 -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.
SummaryA role for the 0~4-integrin (OL4~ 1 or 0t.4~7) , has been imphcated in the recruitment of peripheral blood mononuclear cells (PBMCs) to sites of inflammation. However, the adhesive interactions (i.e., tethering, rolling, and adhesion) mediated by the ot4-integrin have not been characterized in vivo. The objective of this study was to establish a model wherein postcapillary venules were chronically inflamed, and then use intravital microscopy to identify the adhesive interactions mediated by the ot4-integrin in vivo. Between 4 and 20 d after immunization with Mycobacterium butyricum, animals developed a systemic vasculitis characterized by large increases in the numbers of rolling and adhering leukocytes within mesenteric venules. The selectins could only account for "50% of the leukocyte rolling whereas the remaining cells rolled exclusively via the ot4-integrin. Anti-or 4 therapy also ehminated the increase in leukocyte adhesion observed in this model, whereas selectin therapies and an anti-CD18 ([32-integrin) monoclonal antibody (mAb) did not reduce adhesion. A serum against polymorphonuclear leukocytes (PMNs) was used to confirm that a significant proportion of rolling cells, and most of the adhering cells were PBMCs. Sequential treatment with anti-PMN serum and the anti-cq mAb demonstrated that ot4-dependent rolling was distinct from PMN rolling populations. Initial leukocyte tethering via the ot4-integrin could not be demonstrated in this model, whereas L-selectin did support leukocyte tethering. These data suggest that the o~4-integrin can mediate both rolling and adhesion in the multistep recruitment of PMBCs in vivo, and these interactions occur independently of the selectins and [32-integrins. There is a wealth of information describing the recruitment of PMNs from the mainstream of blood to sites ofextravascular injury. This multistep process is initiated by the tethering of PMNs to the endothehum, followed by weak transient adhesive interactions manifested as leukocyte rolling, leading ultimately to firm adhesion of PMNs to the vessel wall (1-4). If an appropriate chemotactic stimulus is present, firm adhesion then allows leukocytes to transmigrate across the vessel wall to sites of potential injury (5, 6). Different mechanisms appear to mediate PMN rolling and adhesion; rolling is dependent on selectins expressed on endothehum (P-selectin, E-selectin) and leukocytes (L-selectin), whereas firm adhesion is dependent on the binding of leukocyte integrins (CDll/CD18) to their ligands (intercellular adhesion molecule 1 [ICAM-1], etc.) found on endothehal cells (3,4,7,8). Intravital microscopy has played a critical role in establishing the molecular mechanisms underlying PMN rolling and adhesion as it provides a window for the direct observation of PMN behavior within intact postcapillary venules. However, because of a lack of chronic inflammatory models amenable to intravital microscopy, there is very htde information regarding the adhesive mechanisms that regulate the influx of other leukocytes su...
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 polyinosinic⅐polycytidylic 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 polyinosinic⅐polycytidylic 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 lungderived 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 sug- IntroductionMast cells are long-lived resident tissue cells found close to blood vessels, and are numerous at sites in close proximity to the external environment such as the skin and airways (reviewed in Galli et al 1 and Metz and Maurer 2 ). From these strategic locations they can quickly recognize and respond to invading pathogens. They are also relatively resistant to ultraviolet (UV) and gamma irradiation. [3][4][5] Upon activation, mast cells produce a wide array of mediators, including granule-associated products, such as histamine, and preformed and de novo synthesized cytokines, chemokines, and lipid mediators. They can activate and recruit effector cells, including eosinophils, 6 neutrophils, 7 and monocytes. 8 Their role in innate immune responses to bacterial infections has been clearly delineated, however their involvement in viral infections is not well understood. Mast cells express Toll-like receptor 3 (TLR3), which recognizes viral double-stranded RNA (dsRNA), 9 and they can produce type I interferons when activated through this receptor. 10 Studies examining the permissiveness of mast cells to viruses show that they can be infected by, and respond to, dengue virus, 11,12 HIV, 13,14 and respiratory syncytial virus. 15 Human mast cells produce the chemokines CCL3, CCL4, and CCL5 when infected with dengue virus, 16 and mouse mast cells produce CCL4 and CCL5 when infected with Newcastle disease virus, all of which are known natural killer (NK) cell and T-cell chemoattractants. 17 NK cells are large granular lymphocytes that can kill virally infected cells, and are crucial for the clearance of viruses during infections (reviewed in Lodoen and Lanier 18 ). The chemokines and chemokine receptors necessary for the infiltration of NK cells into virally infected tissues have recently begun to be uncover...
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. Invest. 1993. 91:577-587.)
Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.
Leukocytes, particularly neutrophils, have been implicated in ischemic-reperfusion organ injury (IRI). However, their role in kidney IRI is controversial. Leukocytes express the adhesion molecules CD11/CD18 on their surface, which mediate many functions that can lead to tissue damage. To determine the role of CD11a and CD11b in IRI in the kidney, uninephrectomized Sprague-Dawley rats were pretreated with monoclonal antibodies (MAbs) directed against CD11a and CD11b or control MAbs. The serum creatinine (SCr), complete blood count, and kidney histopathological damage scores (PDS) (scale: 0-4) were assessed prior to and 24 h after 60 min of ischemia. Mean SCr 24 h after ischemia was significantly decreased in the anti-CD11a- and -CD11b-treated group compared with the control MAb-treated group (2.5 +/- 0.3 mg/dl vs. 3.4 +/- 0.2 mg/dl, P < 0.05). PDS were also reduced in the CD11a and CD11b group compared with controls (2.7 +/- 0.2 vs. 3.5 +/- 0.1, P < 0.001). These data show that the CD11/CD18 leukocyte adhesion pathway plays a role in mediating ischemic acute renal failure in rats.
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