Molecular mechanisms of zinc-dependent leukocyte adhesion involving the urokinase receptor and beta2-integrins. (65/9769)

The trace element Zinc (Zn2+) has been implicated as a mediator in host defense, yet the molecular basis for its extracellular functions remains obscure. Here, we demonstrate that Zn2+ can induce the adhesion of myelomonocytic cells to the endothelium, as well as to the provisional matrix proteins vitronectin (VN) and fibrinogen (FBG), which are pivotal steps for the recruitment of leukocytes into inflamed/injured tissue. Physiologic concentrations of Zn2+ increased the urokinase receptor (uPAR)-mediated adhesion of myelomonocytic cells to VN, whereas other divalent cations had smaller effects. Zn2+-induced cell adhesion to VN was abolished by cation chelators such as 1-10-phenanthroline, as well as by plasminogen activator inhibitor-1 (PAI-1) and a monoclonal antibody (MoAb) against uPAR. These characteristics could be recapitulated with a uPAR-transfected cell line emphasizing the specificity of this receptor system for Zn2+-dependent cell adhesion. Like urokinase (uPA), Zn2+ increased the binding of radiolabeled VN to uPAR-expressing cells, as well as the interaction of VN with immobilized uPAR in an isolated system. Moreover, Zn2+ enhanced leukocytic cell adhesion to FBG and endothelial cell monolayers by activating beta2-integrins. Instead of the direct beta2-integrin activation through the divalent cation binding site, Zn2+-induced integrin activation was mediated via uPAR, a crucial regulator of this system. The present study uncovers for the first time Zn2+-mediated cell adhesion mechanisms that may play a crucial role in modulating leukocyte adhesion to vessel wall components.  (+info)

Survival of donor leukocyte subpopulations in immunocompetent transfusion recipients: frequent long-term microchimerism in severe trauma patients. (66/9769)

We recently reported detection of a transient increase in circulating donor leukocytes (WBCs) in immunocompetent recipients 3 to 5 days posttransfusion (tx) (Blood 85:1207, 1995). We have now characterized survival kinetics of specific donor WBC subsets in additional tx populations. Eight female elective surgery patients (pts) were sampled pre-tx and on days 1, 3, 5, 7, and 14 post-tx. Ten female trauma pts transfused with a total of 4 to 18 U of relatively fresh red blood cells were sampled up to 1.5 years post-tx. WBC subsets from frozen whole blood were isolated using CD4, CD8 (T cell), CD15 (myeloid), and CD19 (B cell) antibody-coated magnetic beads. Donor WBCs were counted by quantitative polymerase chain reaction (PCR) of male-specific sex determining region (SRY) sequences. PCR HLA typing and mixed leukocyte reaction (MLR) between recipient and donor WBCs were performed on two of the trauma tx recipients who had long-term chimerism of donor cells post-tx. In 6 of 8 female surgery pts, circulating CD4(+) male donor cells peaked at day 3 or 5 (0.01 to 1 cell/microL), followed by clearance by day 14. In 7 of 10 female trauma pts, we observed multilineage persistence of male donor WBCs (CD4, CD8, CD15, CD19) for 6 months to 1.5 years post-tx at concentrations of 10 to 100 cells/microL. In 2 trauma recipients studied, MLR showed no, or very low, response to WBC of the single donor implicated as the source of microchimerism by HLA typing. Establishment of long-term multilineage chimerism in trauma recipients is probably caused by engraftment of donor stem cells and mutual tolerance between recipient and donor leukocytes. A better understanding of factors determining clearance versus chimerism of transfused leukocytes is critical to prevention of alloimmunization and transfusion-induced graft-versus-host disease, and, potentially, to induction of tolerance for transplantation.  (+info)

Supernatants from co-cultured endothelial cells and syncytiotrophoblast microvillous membranes activate peripheral blood leukocytes in vitro. (67/9769)

There is evidence for both endothelial cell and peripheral blood leukocyte (PBL) activation in pre-eclampsia. Syncytiotrophoblast microvillous membranes (STBM) are shed in greater quantities from the placenta in pre-eclampsia, disrupt cultured endothelial cells in vitro and may be the immediate cause of the maternal syndrome. The aim of this study was to determine if endothelial cells co-cultured with STBM release factors that can activate PBL in vitro. Flow cytometry was used to measure changes in intracellular free ionized calcium ([Ca2+]i), pH (pHi) and reactive oxygen species (iROS) as indices of leukocyte activation. PBL from male non-pregnant donors was exposed to supernatants from human umbilical vein endothelial cells (HUVEC) cultured with STBM. The time course of changes in [Ca2+]i, pHi and iROS was determined and compared with appropriate control measurements. The test supernatants caused significant activation of granulocytes and monocytes in terms of increases in [Ca2+]i and falls in pHi and release of iROS. Lymphocytes responded only with respect to increases in iROS. The results define a possible mechanism for the activation of PBL in pre-eclampsia, as being secondary to endothelial cell activation caused by circulating STBM shed in excess amounts from the placenta.  (+info)

Leukocyte populations, hormone receptors and apoptosis in eutopic and ectopic first trimester human pregnancies. (68/9769)

The implantation of trophoblast cells at extrauterine sites still results in decidualization. The objective of the present study was to compare decidualization at eutopic and ectopic implantation sites. Tissues from women undergoing elective termination of uterine pregnancy and from women with ectopic pregnancy were used to detect the presence of cells important for the maintenance of pregnancy, such as BCL-2+, CD56+, CD3+, CD8+ and CD68+ cells, and the presence of oestrogen (ER) and progesterone receptors (PR) by immunohistochemistry. In-situ detection of fragmented DNA was performed to identify apoptotic cells. The percentage of CD3+ cells among all immunocompetent cells in the tubal epithelium was 46.6% (39.9% of CD3+ were also CD8+); the other 53.4% were CD68+ cells. CD56+ cells were undetectable in ectopic decidua at the feto-maternal interface in ectopic tissue. In uterine decidua, we found 29.9% CD3+ cells (2.2% of CD3+ were CD8+), 51.6% CD56+ cells and 18.5% CD68+ cells. The ratio of BCL2+ to CD3+ cells in ectopic pregnancy was 0.41. In uterine pregnancy, the ratio of BCL-2 to CD3 was 0.44 and 0.39 for CD56. Tissues from both ectopic and uterine pregnancies were positive for PR. Fewer apoptotic cell bodies were present in ectopic pregnancy. The use of tissue obtained from ectopic pregnancy may become an excellent model to identify the mechanism of trophoblast invasion in eutopic pregnancies.  (+info)

Effects of polysaccharide fucoidin on cerebrospinal fluid interleukin-1 and tumor necrosis factor alpha in pneumococcal meningitis in the rabbit. (69/9769)

The inflammatory response in bacterial meningitis is mediated by cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1), which are produced in the subarachnoid space by different cells, e.g., leukocytes, astrocytes, and microglia. The recruitment of leukocytes into the cerebrospinal fluid (CSF) has been shown to contribute to the neurological damage in this disease, a process which could be enhanced by treatment with antibiotics. In this study, we have used a rabbit meningitis model for two sets of experiments with intracisternal (i.c.) injections of Streptococcus pneumoniae. First, pneumococcal cell wall (PCW) components were injected i.c., inducing an inflammatory response with pleocytosis and increased levels of CSF TNF-alpha) and IL-1 at 6 and 12 h after PCW injection. Treatment with fucoidin, known to inhibit leukocyte rolling, abolished pleocytosis and inhibited the release of TNF-alpha and IL-1. In the second experiment, live pneumococcal bacteria were injected i.c. and treatment with one dose of ampicillin (40 mg/kg of body weight intravenously) was given 16 h after induction of meningitis, causing a sevenfold increase in CSF leukocytes over a 4-h period. CSF IL-1 levels at 16 h were high but did not increase further at 20 h. Also, CSF TNF-alpha levels were high at 16 h and tended to increase at 20 h. Fucoidin treatment prevented the antibiotic-induced increase of CSF leukocytes but had no effect on the TNF-alpha and IL-1 levels. Taken together, fucoidin reduced CSF TNF-alpha and IL-1 levels in acute bacterial meningitis induced by PCW fragments but had no effect later in the course of the disease, when live bacteria were used and an inflammatory increase was caused by a dose of antibiotics.  (+info)

Human opsonins induced during meningococcal disease recognize outer membrane proteins PorA and PorB. (70/9769)

Human opsonins directed against specific meningococcal outer membrane structures in sera obtained during meningococcal disease were quantified with a recently developed antigen-specific, opsonin-dependent phagocytosis and oxidative burst assay. Outer membrane vesicles (OMVs) and PorA (class 1) and PorB (class 3) proteins purified from mutants of the same strain (44/76; B:15:P1.7. 16) were adsorbed to fluorescent beads, opsonized with acute- and convalescent-phase sera from 40 patients with meningococcal disease, and exposed to human leukocytes. Flow cytometric quantitation of the resulting leukocyte phagocytosis products (PPs) demonstrated that disease-induced serum opsonins recognized meningococcal OMV components and both porins. The PPPorA and PPPorB values induced by convalescent-phase sera correlated positively with the PPOMV values. However, the PPPorB values were higher than the PPPorA values in convalescent-phase sera (medians [ranges] of 754 [17 to 1,057] and 107 [4 to 458], respectively) (P < 0.0001) and correlated positively with higher levels of immunoglobulin G against PorB than against PorA as evaluated by enzyme-linked immunosorbent assay. Extensive individual variations in the anti-OMV and antiporin serum opsonic activities between patients infected by serotypes and serosubtypes homologous and heterologous to the target antigens were observed. Simultaneously measured oxidative burst activity correlated with the opsonophagocytosis, an indication that both of these important steps in the in vitro phagocytic elimination of meningococci are initiated by opsonins directed against OMV components, including PorA and PorB. In conclusion, human patient opsonins against meningococcal OMV components and in particular PorB epitopes were identified by this new method, which might facilitate selection of opsonin-inducing meningococcal antigens for inclusion in future vaccines.  (+info)

Endothelial expression of VCAM-1 in experimental crescentic nephritis and effect of antibodies to very late antigen-4 or VCAM-1 on glomerular injury. (71/9769)

The migration of leukocytes into glomeruli in crescentic glomerulonephritis is fundamental to pathogenesis, and offers important therapeutic opportunities. We addressed the importance of VCAM-1, and its leukocyte ligand very late antigen-4 (VLA-4), in such leukocyte migration. In a rat model of nephrotoxic nephritis, glomerular expression of VCAM-1, studied by immunohistochemistry, was up-regulated by day 6 of nephritis. To quantify kidney endothelial VCAM-1 expression, a differential radiolabeled mAb technique was used, which demonstrated that protein expression was not up-regulated by day 2 of nephritis, but rose threefold between days 2 and 5, and remained elevated until at least day 28. An in vivo study was then performed, using blocking mAbs to either VCAM-1 or VLA-4, starting mAb treatment on the day prior to disease induction, and continuing until animals were sacrificed at day 7. mAbs to VLA-4 significantly attenuated renal injury (albuminuria, glomerular fibrinoid necrosis, and crescent formation), but mAbs to VCAM-1 had no significant effect. Surprisingly, the number of leukocytes within glomeruli was unaffected by anti-VLA-4 mAb therapy, despite the reduction in renal injury. Paradoxically, classical markers of macrophage activation were increased in the anti-VLA-4- and anti-VCAM-1-treated animals. This study demonstrates that kidney endothelial VCAM-1, in contrast to ICAM-1, is not up-regulated by day 2 of nephrotoxic nephritis, and plays little part in early leukocyte influx into glomeruli. However, VLA-4 is an important mediator of glomerular injury, operating after transendothelial leukocyte migration, and presumably binding to alternate ligands within the kidney.  (+info)

Relationship between chimerism and tolerance in a kidney transplantation model. (72/9769)

The persistence of donor leukocytes in recipients of organ allografts has been associated with long-term graft acceptance. However, it remains unclear whether this peripheral donor cell microchimerism plays an active role in graft acceptance or is simply a consequence of the maintenance of sufficient immunosuppression to avoid rejection. A model of kidney transplantation between swine leukocyte Ag (SLA)-matched miniature swine, in which tolerance can be established with or without immunosuppressive treatment, has been used to study the correlation between donor leukocyte chimerism and kidney graft acceptance. SLA-identical kidney transplants were performed from animals positive for an allelic pig leukocyte Ag to animals negative for this marker. SLA-identical kidney transplant recipients given a 12-day course of cyclosporine (CyA) (n = 3) became tolerant, showing stable serum creatinine levels (1-2 mg/dl) after cessation of CyA treatment. Donor cell chimerism (0.2-0.7%) was present by FACS in all three animals with peak levels detected at 3 wk. Two control animals receiving SLA-identical kidney grafts without CyA also showed stable serum creatinine levels and became tolerant. However, in neither of these animals could donor leukocytes be detected in the peripheral blood beyond 1 wk following transplantation. In one additional control animal, ureteral obstruction occurred at day 10, and was associated with additional peripheral chimerism, presumably related to inflammation rather than to immune status. These results indicate that the persistence of donor cell chimerism is not a requirement for the maintenance of tolerance to organ allografts in this model.  (+info)