Henry Beekhuizen

Monocyte adherence to human vascular endothelium

An acute inflammatory response requires that circulating monocytes bind to and migrate across the endothelium of the vessel wall to gain access to inflamed sites. Various mediators of inflammation ‐ cytokines and chemoattractants ‐ have been shown to initiate and regulate the margination and extravasation of monocytes. This review summarizes evidence that the mechanism underlying the initial adhesion of monocytes to normal and cytokine‐stimulated endothelial cells and their subsequent transendothelial migration are successive events in monocyte‐endothelial cell interaction. Special emphasis is given to the current knowledge of the contribution of adhesion molecules belonging to the family of 13i‐ and 132‐integrins, the immunoglobulin supergene family, and the selectins to such cellular interaction. The sequence of events that allow monocytes to attach to, migrate over and finally pass the endothelium is discussed in detail.

Growth Characteristics of Cultured Human Macrovascular Venous and Arterial and Microvascular Endothelial Cells

The morphological and growth characteristics of human macrovascular endothelial cells (ECs) from venous and arterial umbilical cord vessels and microvascular ECs from foreskin were compared during cultivation. By means of time-lapse microcinematography and phase-contrast microscopy, differences in cell morphology and migratory activity between the different types of ECs were found. Growth characteristics were dependent on the type of EC, the nature of the substrates on which the ECs were grown and the presence of growth factors. For all types of ECs optimal growth and formation of a monolayer were observed when the ECs were cultured on fibronectin or gelatin substrates in the presence of EC growth factor and heparin. Under these conditions confluent cultures of macrovascular ECs reached maximal cell densities of 1,400-1,900 ECs/mm2, whereas microvascular ECs reached maximal cell densities of about 700-900 ECs/mm2. The cell cycle times calculated from the population-doubling time and the stathmokinetic index, respectively, amounted to 63 and 83 h for microvascular ECs, 33 and 35 h for venous macrovascular ECs, and 29 and 35 h for arterial macrovascular ECs.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

Monocytes augment bacterial species- and strain-dependent induction of tissue factor activity in bacterium-infected human vascular endothelial cells.

ABSTRACT In bacterial endocarditis (BE), intravascular infection withStaphylococcus aureus, Streptococcus sanguis, or Staphylococcus epidermidis can lead to formation of a fibrin clot on the inner surface of the heart and cause heart dysfunction. The events that start the coagulation in the early stage of the disease are largely unknown. We have recently shown that human endothelial cells (EC) upon binding and internalization ofS. aureus, but not S. sanguis orS. epidermidis, express tissue factor (TF)-dependent procoagulant activity (TFA). The present study shows that infection of EC with these three pathogens induces surface expression of intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) and monocyte adhesion. Subsequent coculture of these cells synergistically enhanced TFA, which was exclusively dependent on TF molecules that were expressed on EC during coculture. TFA induction required direct contact between monocytes and bacterium-infected EC, but the signals for this response were not generated by the binding of monocytes through their β2- or α4-integrins to ICAM-1 or VCAM-1, respectively, on infected EC. The mechanism by which monocytes induce TFA in bacterium-infected EC was partly mediated by the proinflammatory cytokine interleukin-1 produced by the cells during coculture. Endogenous tumor necrosis factor alpha was not involved. This modulating effect of monocytes on species- and strain-dependent TFA of bacterium-infected EC supports our hypothesis that in an early stage in the pathogenesis of BE, as well as other intravascular infections that lead to detrimental fibrin formation, the coagulation cascade can be activated on the surfaces of EC as a consequence of specific interactions between pathogenic bacteria, EC, and monocytes.

Protection and humoral immune responses against Bordetella pertussis infection in mice immunized with acellular or cellular pertussis immunogens

In the present study, protection against Bordetella pertussis infection and humoral immunological responses in mice has been assessed upon immunization with custom-made acellular pertussis vaccines (ACVs) and whole-cell pertussis vaccine (WCV). Mice were immunized, next intranasally infected with B. pertussis and during 14 days the number of bacteria in the trachea and lungs and the level of serum antibodies were determined. ACV contained five immunogens, filamentous hemagglutinin, pertactin, fimbriae serotypes 2 and 3, and chemically detoxified pertussis toxin (PMC-5), or three immunogens, filamentous hemagglutinin, pertactin, and genetically detoxified (BC-3) or chemically detoxified pertussis toxin (SKB-3). Immunization with a high or low dose of ACV or WCV resulted in significant protection against B. pertussis, with differences in the degree of protection between the vaccines. The lowest protection was found with a low dose of SKB-3 and WCV. The pattern of cytokine production by spleen cells of immunized, non-infected, mice indicated that T-helper 1 cells are activated by vaccination with WCV, and T-helper 1 and T-helper 2 cells are involved in the immune response upon vaccination with ACVs. Each vaccine stimulated the production of IgG, but not IgA, antibodies. In mice immunized with ACV, elimination of B. pertussis from trachea and lungs correlated significantly with the titre of IgG1, but not IgG2a, antibodies.

Altered Gene Expression in Staphylococcus aureus upon Interaction with Human Endothelial Cells

ABSTRACT Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureusto human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction betweenS. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureuswas used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells

The Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA sub-domains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604-877) or A4(+16) (aa 432-559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.

Monocytes Maintain Tissue Factor Activity after Cytolysis of Bacteria-Infected Endothelial Cells in an In Vitro Model of Bacterial Endocarditis

Intravascular infection with Staphylococcus aureus, Staphylococcus epidermidis, or Streptococcus sanguis can initiate fibrin formation on endocardial tissue, causing bacterial endocarditis. The ability of these bacteria to injure intact endothelial cells (ECs) and to aggravate tissue factor (TF)-dependent coagulation in the presence of blood leukocytes was investigated. Cytolysis of ECs occurred after infection with S. aureus and, with membrane-bound monocytes or granulocytes present, also after infection with S. sanguis or S. epidermidis. Monocytes that subsequently bound to the resultant bacteria-infected subcellular EC matrix (ECM) elicited TF mRNA, TF antigen, and TF activity (TFA). This was most pronounced in ECM prepared after the cytolysis of ECs by infection with S. aureus or S. epidermidis. We demonstrate that monocytes continue and intensify fibrin formation after lysis of bacteria-infected ECs, which suggests that, during the course of intravascular infection, early fibrin formation shifts from being mediated by EC-derived TFA to being mediated by TFA of monocytes bound to bacteria-infected ECM.

Gamma Irradiation or CD4-T-Cell Depletion Causes Reactivation of Latent Salmonella enterica Serovar Typhimurium Infection in C3H/HeN Mice

ABSTRACT Upon infection with Salmonella, a host develops an immune response to limit bacterial growth and kill and eliminate the pathogen. Salmonella has evolved mechanisms to remain dormant within the body, only to reappear (reactivate) at a later time when the immune system is abated. We have developed an in vivo model for studying reactivation of Salmonella enterica serovar Typhimurium infection in mice. Upon subcutaneous infection, C3H/HeN (Ityr) mice showed an increase in bacterial numbers in livers and spleens, which reached a peak on day 19. After full recovery from the infection, these mice were irradiated or depleted of CD4+ T cells. The mice displayed a secondary infection peak in livers and spleens with a course similar to that of the primary infection. We concluded that CD4+ T cells are involved in active suppression of S. enterica serovar Typhimurium during latency. The role of CD4+ T cells during primary infection with S. enterica serovar Typhimurium is well established. This is the first study to describe a role of CD4+ T cells during the latent phase of S. enterica serovar Typhimurium infection.

Gamma Interferon Confers Resistance to Infection with Staphylococcus aureus in Human Vascular Endothelial Cells by Cooperative Proinflammatory and Enhanced Intrinsic Antibacterial Activities

ABSTRACT Vascular endothelium is an exposed target in systemic endovascular Staphylococcus aureus infections. We reported earlier that the proinflammatory and procoagulant activities of primary human umbilical vein endothelial cells (ECs) after binding and ingestion of S. aureus organisms provide the cells effective means for leukocyte-mediated bacterial elimination. Expanding on this, we now show that these ECs exhibit a modest intrinsic capacity for eliminating intracellular S. aureus that was influenced by cytokines relevant to S. aureus infections. Using various EC infection assays, we showed that gamma interferon (IFN-γ), applied to cultures of ECs prior to or after infection with S. aureus, markedly reduced the level of infection, illustrated by lower percentages of S. aureus-infected ECs and less intracellular bacteria per infected cell. IFN-γ-activated ECs had unaltered abilities to bind S. aureus and processed ingested bacteria by a seemingly conventional phagocytic pathway. IFN-γ treatment rescued EC monolayers from severe injury by virulent clinical S. aureus strains or excessive bacterial numbers. Mechanistically, IFN-γ controls S. aureus infection via IFN-γ receptor, most likely through stimulation of intrinsic endothelial antibacterial mechanisms but independent of processes that deprive bacteria of intracellular l-tryptophan or iron. The antibacterial activity of IFN-γ-stimulated ECs coincided with sustained or slightly elevated endothelial proinflammatory responses that supported monocyte recruitment. In conclusion, we identify IFN-γ as a potent regulatory Th1 cytokine possessing exclusive abilities to augment intrinsic antistaphylocccal effector mechanisms in human ECs without ablating the S. aureus-induced proinflammatory EC responses and, as such, coordinating a protective efficacy of ECs against blood-borne S. aureus infection.