Antigen presentation by macrophages is enhanced by the uptake of necrotic, but not apoptotic, cells

The aim of this study was to determine whether phagocytosis of necrotic or apoptotic cells affects antigen presentation by murine bone marrow-derived macrophages. After uptake of necrotic neutrophils, macrophages were able to stimulate significantly higher T cell proliferation in vitro against both the recall antigen albumin and the mitogen concanavalin A. No such effect was seen following phagocytosis of apoptotic neutrophils. Flow cytometry revealed that, within 4h of ingestion, macrophages that had taken up the necrotic cells expressed higher levels of CD40 than those that had phagocytosed apoptotic cells. Macrophage cultures pulsed with apoptotic, but not necrotic, neutrophils contained higher levels of transforming growth factor beta1, but lower concentrations of tumour necrosis factor alpha, compared to untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic neutrophils co-stimulate T cells with greater efficiency due to rapid CD40 up-regulation, whereas those that have ingested apoptotic cells are not only ineffective in co-stimulation, but also secrete inhibitory cytokine.     

Antigen processing and presentation by macrophages

The classical macrophage is one of the most important cells involved in presenting antigen to helper T cells, because of its ability to regulate its expression of Ia molecules and to encounter and process particulate and soluble antigens. We have summarized in this report studies examining the handling by macrophages of two different antigens, the bacteria Listeria monocytogenes and the protein hen egg white lysozyme (HEL). The purpose was to identify potential sources of immunogenic peptides. Presentation of Listeria required an intracellular processing stage sensitive to lysosomotropic drugs. The Listeria required internalization and processing, after which immunogenic molecules were recognized by T cells on the macrophage surface. Metabolic studies showed that Listeria-derived peptides were released by macrophages that had phagocytosized the bacteria. The release of these peptides was a temperature-dependent process, unaffected by inhibiting lysosomal catabolism by treatment with chloroquine. Listeria-derived peptides were also detected on the surface of the macrophage. These peptides behaved like integral membrane proteins, some of which persisted for at least 24 hr at the macrophage surface. When tested for immunogenicity, the released peptides were very weakly immunogenic. The membrane-associated peptides alone could not stimulate Listeria-specific T cells, but could be reprocessed by additional macrophages and subsequently stimulate the T cells. A defined antigen system using HEL-specific T-cell hybridomas was used to examine the processing of HEL. Presentation of HEL required a choloroquine-sensitive intracellular processing stage. In examining two T-cell hybridomas, a differential requirement for antigen processing was determined. The immunogenicity of released HEL from HEL-pulsed macrophages was also tested, and no “processed” antigen was detected capable of directly stimulating T cells; however, native HEL was released from the macrophage and could be processed and presented by other macrophages.     

Mechanisms of resistance of Porphyromonas gingivalis to killing by serum complement

The complement system plays an important role in the host defense against infection, and the formation of the terminal complement complex on the bacterial surface has been shown to be particularly important in killing of gram-negative bacteria. The gram-negative periodontal pathogen Porphyromonas gingivalis is resistant to complement killing, and possible mechanisms suggested for this resistance include protease production and capsule formation. In this study, P. gingivalis Arg- and Lys-gingipain deletion mutants and polysaccharide synthesis deletion mutants have been used to investigate these hypotheses. When Arg- and Lys-gingipain protease mutants were incubated in 20% normal human serum, deposition of complement components on the cell surface was significantly increased compared to that for the wild-type organism. However, despite the increased deposition, the protease mutants maintained resistance to killing and their viability was equal to that seen with heat-inactivated serum. Similar data were obtained when the wild-type organism was treated with gingipain protease inhibitors. K-antigen expression mutants were also resistant to killing. However, mutants which no longer synthesized a surface anionic polysaccharide (APS) (a phosphorylated branched mannan) were extremely sensitive to serum killing. These mutants lack the organized dense glycan surface layer present on the parent strain on the basis of electron microscopy. We conclude that the production of APS at the surface of P. gingivalis rather than Arg- and Lys-gingipain synthesis is the principal mechanism of serum resistance in P. gingivalis.     

Antigen presentation by keratinocytes induces tolerance in human T cells

Antigen recognition by interleukin 2 (IL 2)-producing T lymphocytes can lead to two distinct outcomes, depending on the nature of the antigen-presenting cell. Recognition of antigen presented by specialized antigen-presenting cells leads to T cell activation; in contrast, antigen presentation by cells which lack "accessory function" can lead to a state of specific nonresponsiveness, which is characterized by a failure to produce IL 2. We have shown in this study that co-culture of an HLA-DR1/4-restricted, influenza hemagglutinin-specific T cell clone with a specific peptide presented by interferon-gamma-induced DR4-expressing keratinocytes causes tolerance induction. This effect was DR restricted, in that it required pre-incubation of the T cell clone with keratinocytes expressing an appropriate DR type (DR4Dw14). The induction of T cell tolerance was also antigen specific; no inhibition resulted from pre-incubation of the clone with an irrelevant peptide. Furthermore cell to cell contact appeared to be necessary, and the addition of supernatant from interferon-gamma-induced keratinocytes did not cause any inhibition. This phenomenon may have relevance to the immunogenicity of transplanted cultured keratinocytes and to the effects of major histocompatibility complex class II induction on non-bone marrow-derived cells. Presentation of tissue-specific autoantigens by cells such as keratinocytes may provide a mechanism of avoiding, rather than stimulating, autoimmune reactions in the context of a local inflammatory response.     

In vivo exposure to Porphyromonas gingivalis up-regulates nitric oxide but suppresses tumour necrosis factor-alpha production by cultured macrophages.

The present study was designed to test whether the functional response of mouse macrophages elicited by chronic exposure to bacteria will be different from that of cells elicited by a non-bacterial irritant. Macrophage elicitation was conducted by Porphyromonas gingivalis, a major periodontal pathogen, in comparison to a standard elicitation by thioglycollate (TG). We measured lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) secretion by the elicited macrophages, and the expression of inflammatory cytokines in the whole elicited cell population. In addition, we tested the response of TG-elicited macrophages to pretreatment with P. gingivalis LPS in vitro. Mouse peritoneal macrophages were harvested 4 days after intraperitoneal injection of TG or heat-killed P. gingivalis. TG-elicited macrophages produced undetectable levels of TNF-alpha and approximately 0.5 microM of NO. The stimulation of the macrophages with LPS resulted in the secretion of NO and TNF-alpha in a dose-dependent manner. The P. gingivalis-elicited macrophages produced basal levels of approximately 5 microM NO, but TNF-alpha was not detectable. LPS stimulation of these cells further increased the secretion of NO eightfold while TNF-alpha remained undetectable. The NO secretion by P. gingivalis-elicited cells was significantly higher than that by TG-elicited cells. Examination of cytokine expression in the whole elicited cell population revealed that both P. gingivalis-elicited cells and TG-elicited cells expressed messenger RNA for interleukin-2 (IL-2), TNF-alpha and interferon-gamma (IFN-gamma), but not for IL-4. IL-6 was expressed in P. gingivalis-elicited cells only. Pretreatment of TG-elicited macrophages with P. gingivalis LPS for 24 hr prior to a second LPS challenge resulted in down-regulation of TNF-alpha secretion and up-regulation of NO secretion, a response similar to that seen in P. gingivalis-elicited peritoneal macrophages. The results suggest that the in vivo exposure of resident macrophages to P. gingivalis induces functional changes in peritoneal macrophages. These changes might be due to the effect of P. gingivalis LPS.     

Intracellular trafficking and killing of Streptococcus pneumoniae by human alveolar macrophages are influenced by opsonins

Human alveolar macrophages (HAM) are the major resident phagocytic cells of the gas-exchanging areas of the lung. Following contact with macrophages, bacteria enter phagosomes, which gradually acquire the characteristics of terminal phagolysosomes, with incorporation of lysosome-associated membrane protein (LAMP). We measured the binding of type 1 Streptococcus pneumoniae to the surface of HAM and then measured subsequent internalization and phagosomal incorporation of LAMP-1 under various opsonic conditions. Opsonization with serum containing immunoglobulin resulted in significantly greater binding of pneumococci to HAM compared with opsonization with immunoglobulin G (IgG)-depleted serum containing complement, which in turn resulted in marginally increased binding over that observed in the absence of opsonization. Internalization of opsonized S. pneumoniae gradually increased to a maximum of 20% of bound bacteria by 120 min of warm incubation, with 20% of internalized pneumococci being localized within LAMP-containing compartments by 80 min. Internalization of opsonized S. pneumoniae by HAM correlated with a reduction of bacterial viability. When inocula were adjusted so that pneumococcal binding under different conditions was equalized, subsequent internalization, trafficking to LAMP-containing compartments, and reduction of bacterial viability were less efficient in the absence of opsonization than that observed following opsonization with adsorbed or IgG-replete adsorbed serum. Once bound to the surface of HAM, pneumococci opsonized with adsorbed serum with or without IgG were internalized, processed, and killed equally well. In conclusion, binding, intracellular trafficking, and killing of S. pneumoniae by HAM are each significantly increased by opsonization with serum containing immunogloblin and/or complement.     

Human endothelial cell responses to endotoxin are dramatically enhanced by human blood

Vascular endothelia activated by endotoxin (lipopolysaccharide, LPS) or cytokines actively participate in local and systemic inflammation such as those observed during Gram-negative sepsis, by secreting cytokines and chemokines, and upregulating adhesions molecules for blood leukocytes. LPS can directly activate endothelial cells via its interaction, with soluble CD14, but may also activate endothelial cell indirectly through LPS-induced, monocyte-derived cytokines. Here we show that the addition of blood cells to plasma rendered the endothelial cells sensitive to LPS concentrations 1000 times lower than when only plasma was present. We demonstrated that this effect was carried by monocytes and more precisely by soluble factors produced by monocytes, and they were further identified as tumor necrosis factor (TNF) and interleukin-1 (IL-1).     

Role of superoxide dismutase in resistance of Porphyromonas gingivalis to killing by polymorphonuclear leukocytes.

Porphyromonas gingivalis in which the synthesis of superoxide dismutase (SOD) was induced by nitrate or by aeration was rendered resistant to killing by polymorphonuclear leukocytes. SOD purified from either anaerobically maintained or aerated cells also inhibited bacterial killing when added exogenously, and no difference between the effects of the two SODs was observed. These results suggest that SOD may form part of a defense mechanism that helps protect P. gingivalis against killing by polymorphonuclear leukocytes.     

Binding and accumulation of hemin in Porphyromonas gingivalis are induced by hemin.

Although hemin is an essential nutrient for the black-pigmented oral bacterium Porphyromonas gingivalis, the mechanisms involved in hemin binding and uptake are poorly defined. In this study, we have examined the binding of hemin and Congo red (CR) to P. gingivalis whole cells and have defined the conditions for maximal binding. Additionally, the accumulation of hemin by P. gingivalis under growing conditions has been characterized. P. gingivalis A7436 was grown under hemin- or iron-deplete conditions (basal medium [BM] or Schaedler broth with dipyridyl [SBD]) or under hemin- or iron-replete conditions (BM with hemin [BMH] or Schaedler broth [SB]), and hemin and CR binding were assessed spectrophotometrically. Binding of hemin by P. gingivalis whole cells was rapid and was observed in samples obtained from cells grown under hemin- and iron-replete and hemin-deplete conditions but was not observed in cells grown under iron limitation. We also found that P. gingivalis whole cells bound more hemin when grown in BMH or SB than cells grown in BM or SBD. Binding of CR by P. gingivalis A7436 was also enhanced when cells were grown in the presence of hemin or when cells were incubated with hemin prior to CR binding. Hemin binding and accumulation were also assessed using [14C]hemin and [59Fe]hemin under growing conditions. Both [14C]hemin and [59Fe]hemin were accumulated by P. gingivalis, indicating that iron and the porphyrin ring were taken into the cell. Binding and accumulation of hemin under growing conditions were also induced by growth of P. gingivalis in hemin-replete media. Hemin accumulation was inhibited by the addition of KCN to P. gingivalis cultures, indicating that active transport was required for hemin uptake. [14C]hemin binding and accumulation were also inhibited by the addition of either cold hemin or protoporphyrin IX. Taken together, these results indicate that P. gingivalis transports the entire hemin moiety into the cell and that the binding and accumulation of hemin are induced by growth of cultures in the presence of hemin.     

Antigen presenting cells and mechanisms of antigen presentation

In this review we will examine the characteristics of the various cell types which have been shown to present antigens to helper and proliferating T cells and explore what is known about the requirements for antigen presentation by these cells. Cell types to be discussed include mononuclear phagocytes from a variety of tissues as well as nonphagocytic cells such as Langerhans cells and dendritic cells. Special consideration will be given to the most recent group of cells to have demonstrated antigen-presenting capacity, B lymphocytes. Experiments exploring the processing and presentation of antigen by these different cell types will be presented. These results suggest that immunologically relevant antigen is endocytosed and at least partially degraded before proper presentation can occur. The role of molecules synthesized by the presenting cells, such as MHC antigens and cytokines, will be discussed in detail.     

Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b

Antigen presentation and microbial killing are critical arms of host defense that depend upon cargo trafficking into lysosomes. Yet, the molecular regulators of traffic into lysosomes are only partly understood. Here, using a lysosome-dependent immunological screen of a trafficking shRNA library, we identified the Arf-like GTPase Arl8b as a critical regulator of cargo delivery to lysosomes. Homotypic fusion and vacuole protein sorting (HOPS) complex members were identified as effectors of Arl8b and were dependent on Arl8b for recruitment to lysosomes, suggesting that Arl8b-HOPS plays a general role in directing traffic to lysosomes. Moreover, the formation of CD1?antigen-presenting complexes in lysosomes, their delivery to the plasma membrane, and phagosome-lysosome fusion were all markedly impaired in Arl8b silenced cells resulting in corresponding defects in T?cell activation and microbial killing. Together, these results define Arl8b as a key regulator of lysosomal cellular and immunological functions.     

Differential activation of human gingival epithelial cells and monocytes by Porphyromonas gingivalis fimbriae

Humans develop periodontitis in response to challenge by microbial dental plaque. Inflammation begins after perturbation of gingival epithelial cells by subgingival bacteria interacting through pattern-recognition receptors, including the Toll-like receptors (TLR). Porphyromonas gingivalis is a major periodontopathogen that interacts with epithelial cells through its cell surface fimbriae (FimA), leading to colonization and/or invasion. Previous work by our group has established membrane CD14 as an essential coreceptor for TLR2-mediated activation of transfected cell lines by P. gingivalis FimA. We have shown that gingival epithelial cells express TLR2 but not CD14 on their cell surfaces. We thus speculated that P. gingivalis FimA does not readily activate epithelial innate immune responses but rather functions to promote P. gingivalis colonization in the absence of a vigorous FimA-induced response. This hypothesis was verified by the findings that primary human gingival epithelial cells responded poorly to FimA in terms of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha responses, in stark contrast to the marked response to other TLR2 agonists (Pam3Cys, FSL-1) that are not strictly dependent on CD14. On the other hand, CD14-expressing human primary monocytes responded with high levels of the same cytokines to both FimA and the control TLR2 agonists. The gingival epithelial cells failed to respond to FimA even in the presence of exogenously added soluble CD14. These data indicate that the gingival epithelial cell hyporesponsiveness to FimA is attributable to the lack of membrane-expressed but not soluble CD14. In conclusion, P. gingivalis FimA differentially activates human monocytes and epithelial cells, perhaps reflecting different tactics used by P. gingivalis when interacting with different host cell types or a host strategy to limit inflammation.     

Interferon gamma-treated human macrophages display enhanced cytolysis and generation of reactive oxygen metabolites but reduced ingestion upon Fc receptor triggering

Human monocyte-derived macrophages treated with recombinant IFN-gamma (rIFN-gamma) and control cells were assessed for three distinct effector functions, all mediated by Fc receptors. rIFN-gamma-primed macrophage displayed markedly reduced phagocytosis of IgG antibody-coated erythrocytes. In contrast, antibody-dependent cytotoxicity towards IgG-antibody-coated erythrocytes and IgG-antibody-coated erythrocyte-induced generation of reactive oxygen metabolite production were increased. The decreased phagocytosis was observed microscopically, as well as in a spectrometric and a radiometric phagocytosis assay. Evidence is presented that the observed impairment in phagocytosis is not the result of increased extracellular lysis or intracellular catabolism of IgG-antibody-coated erythrocytes and that it is not observed with particles ingested in an Fc receptor-independent manner. Enhanced production of reactive oxygen metabolites was detected most clearly by measurement of luminol-dependent chemiluminescence. Antibody-dependent cellular cytotoxicity was shown to proceed also under conditions impeding phagocytosis, and rIFN-gamma-treated macrophage exerted enhanced antibody-dependent cellular cytotoxicity under these conditions too. In all three assays, functional alterations were optimally expressed after a treatment with 500 U/ml for 46 hr. Analysis at the single-cell level revealed that the IFN-gamma-induced alterations were expressed by all macrophages and not the property of distinct macrophage subpopulations. This and earlier studies suggest that the modulation of Fc receptor-mediated macrophage effector functions by IFN-gamma is in part a post-receptor-binding event.     

Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: Definition of a maturative step

Human CD1⁺ CD14^- dendritic cells (DC) can be derived from CD14⁺ monocytes using granulocyte/monocyte colony-stimulating factor and interleukin (IL)-4. We have previously shown that IL-10 pre-treatment of such DC significantly inhibited their antigen-presenting capacity to CD4⁺ T cell clones. In this study, we further analyze how IL-10 influences antigen presentation. We first investigated whether IL-10 could alter the early stage of antigen presentation, the capture of antigen. This can be mediated by mannose receptor (MR)-mediated endocytosis and by fluid-phase uptake through macropinocytosis. IL-10-treated DC showed an enhancement of both mechanisms of antigen capture, as indicated by the increase of fluorescein isothiocyanate-dextran uptake through MR and lucifer yellow uptake. However, IL-10-treated DC, irradiated or glutaraldehyde-fixed, were less efficient than untreated DC in stimulating mixed leukocyte reaction as well as in inducing the activation of peptide-specific T cell clones, indicating that IL-10 achieves its effects mainly by modifying the cell surface phenotype of DC. HLA class I and II, as well as intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen-3, B7-1, B7-2 and ICAM-3 expression were either significantly increased or essentially unchanged, and the ability to bind the epitope recognized by the T cell clones was also unaffected regardless of IL-10 treatment. Our study also indicates that as-yet unidentified accessory molecules may play an essential role in T cell activation. Thus, the IL-10-treated DC possess an increased capacity to capture antigen, with a concomitant decreased stimulatory activity. Our study suggests that IL-10-treated DC have the characteristics of highly immature DC (high capture ability, low stimulatory potency) and may represent an early maturative step of human DC of monocytic origin.     

Kgp and RgpB, but not RgpA, are important for Porphyromonas gingivalis virulence in the murine periodontitis model

The contributions of three proteinase genes (rgpA, rgpB, and kgp) to the virulence of Porphyromonas gingivalis W50 were investigated in the murine periodontitis model. Mice were orally inoculated with eight doses (1 x 10(10) cells per dose) of rgpA, rgpB, kgp, rgpA rgpB, or rgpA rgpB kgp isogenic mutants, and the level of alveolar bone loss, immune response induced, and number of bacterial cells per half maxilla were compared with those of animals inoculated with wild-type P. gingivalis. The kgp, rgpB, rgpA rgpB, and rgpA rgpB kgp isogenic mutants induced significantly (P < 0.05) less bone loss than the rgpA isogenic mutant and the wild type did, and the virulence of the rgpA isogenic mutant and the wild type were not significantly different. Mice inoculated with the wild type or the rgpA isogenic mutant exhibited significantly (P < 0.01) more P. gingivalis cells per half maxilla than mice inoculated with rgpB, kgp, rgpA rgpB, and rgpA rgpB kgp isogenic mutants or nonchallenged mice did, as determined using real-time PCR. A significant positive correlation was found between the number of P. gingivalis cells detected per half maxilla and the amount of alveolar bone loss induced. Enzyme-linked immunosorbent assay results showed that each isogenic mutant and the wild type induced a predominant P. gingivalis antigen-specific immunoglobulin G3 (IgG3) response. Furthermore, the kgp and rgpA rgpB kgp isogenic mutants induced significantly (P < 0.05) lower IgG3 antibody responses than the responses induced by the wild type or the rgpA, rgpB, and rgpA rgpB isogenic mutants. The results suggest that the order in which the proteinases contribute to the virulence of P. gingivalis in the murine periodontitis model is Kgp > or = RgpB > RgpA.     

Citrullination and Proteolytic Processing of Chemokines by Porphyromonas gingivalis

The outgrowth of Porphyromonas gingivalis within the inflammatory subgingival plaque is associated with periodontitis characterized by periodontal tissue destruction, loss of alveolar bone, periodontal pocket formation, and eventually, tooth loss. Potential virulence factors of P. gingivalis are peptidylarginine deiminase (PPAD), an enzyme modifying free or peptide-bound arginine to citrulline, and the bacterial proteases referred to as gingipains (Rgp and Kgp). Chemokines attract leukocytes during inflammation. However, posttranslational modification (PTM) of chemokines by proteases or human peptidylarginine deiminases may alter their biological activities. Since chemokine processing may be important in microbial defense mechanisms, we investigated whether PTM of chemokines by P. gingivalis enzymes occurs. Upon incubation of interleukin-8 (IL-8; CXCL8) with PPAD, only minor enzymatic citrullination was detected. In contrast, Rgp rapidly cleaved CXCL8 in vitro. Subsequently, different P. gingivalis strains were incubated with the chemokine CXCL8 or CXCL10 and their PTMs were investigated. No significant CXCL8 citrullination was detected for the tested strains. Interestingly, although considerable differences in the efficiency of CXCL8 degradation were observed with full cultures of various strains, similar rates of chemokine proteolysis were exerted by cell-free culture supernatants. Sequencing of CXCL8 incubated with supernatant or bacteria showed that CXCL8 is processed into its more potent forms consisting of amino acids 6 to 77 and amino acids 9 to 77 (the 6-77 and 9-77 forms, respectively). In contrast, CXCL10 was entirely and rapidly degraded by P. gingivalis, with no transient chemokine forms being observed. In conclusion, this study demonstrates PTM of CXCL8 and CXCL10 by gingipains of P. gingivalis and that strain differences may particularly affect the activity of these bacterial membrane-associated proteases.     

Antigen presentation by Leishmania mexicana-infected macrophages: Activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites

Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (T_h1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-γ do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-γ and tumor necrosis factor-α. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.     

Inhibition of Porphyromonas gingivalis adhesion to Streptococcus gordonii by human submandibular-sublingual saliva.

Porphyromonas gingivalis W50 adheres in vitro to biofilms of Streptococcus gordonii G9B. This phenomenon is believed to facilitate the initial colonization of the oral cavity by P. gingivalis and to contribute to the maturation of dental plaque. In this report, we describe the modulating effects of human submandibular-sublingual saliva (HSMSL) on this in vitro model of intergeneric bacterial adhesion (coaggregation). HSMSL inhibited P. gingivalis adhesion to S. gordonii by 50% at a concentration of 57 micrograms of protein per ml. Maximum inhibitory activity was associated with a 43-kDa protein obtained by sequential Sephadex G200 gel filtration and CM52 ion-exchange chromatography of HSMSL. Pools of other column fractions of HSMSL showed no effect or were slightly stimulatory for bacterial adhesion. The binding of radioiodinated column fractions containing the 43-kDa protein by P. gingivalis was accompanied by their rapid enzymatic degradation. Treating P. gingivalis at 60 degrees C for 30 min or with protease inhibitors (phenylmethylsulfonyl fluoride and sodium iodoacetate) reduced adherence to streptococcal biofilms. These treatments did not prevent P. gingivalis from binding soluble HSMSL saliva components, although subsequent proteolysis was nearly eliminated. These observations indicate that surface-associated proteases of P. gingivalis, either independently or in concert with adjacent surface adhesins, interact with surfaces of oral streptococci to facilitate interbacterial adhesion. The adhesion-blocking properties of HSMSL, particularly the 43-kDa protein, may represent an important host defense mechanism in the oral cavity.     

Porphyromonas gingivalis fimbriae proactively modulate beta2 integrin adhesive activity and promote binding to and internalization by macrophages

In monocytes, the fimbriae of the oral pathogen Porphyromonas gingivalis activate cross talk signaling from Toll-like receptor 2 (TLR2) to the beta2 integrin CD11b/CD18, leading to the induction of the high-affinity state of the latter receptor. CD14 plays an important role in this "inside-out" proadhesive pathway by binding fimbriae and facilitating the activation of TLR2 and phosphatidylinositol 3-kinase signaling. In its high-affinity state, CD11b/CD18 mediates monocyte adhesion to endothelial cells and transmigration to sites of infection. We have now shown that P. gingivalis fimbriae function as both an activator and a ligand of CD11b/CD18; thus, fimbriae proactively promote their own binding to monocytes. Indeed, treatments that interfered with fimbria-induced activation of CD11b/CD18 (i.e., blockade of CD14, TLR2, or phosphatidylinositol 3-kinase signaling) also suppressed the cell binding activity of fimbriae, which was largely inducible and CD11b/CD18 dependent. Development of a recombinant inside-out signaling system in Chinese hamster ovary cells confirmed the ability of fimbriae to activate CD14/TLR2 signaling and induce their own CD11b/CD18-dependent binding. Induction of this proadhesive pathway by P. gingivalis fimbriae appeared to take place in lipid rafts. Indeed, methyl-beta-cyclodextrin, a cholesterol-sequestering agent that disrupts lipid raft organization, was found to inhibit the fimbria-induced assembly of CD14/TLR2 signaling complexes and the activation of the high-affinity state of CD11b/CD18. Experiments using macrophages from mice deficient in various pattern recognition receptors indicated that the receptors involved in the inside-out proadhesive pathway (CD14, TLR2, and CD11b/CD18) are important for mediating P. gingivalis internalization within macrophages. It therefore appears that P. gingivalis proactively modulates beta2 integrin adhesive activity for intracellular uptake.     

Hemagglutinin B is involved in the adherence of Porphyromonas gingivalis to human coronary artery endothelial cells

Porphyromonas gingivalis is a periodontopathogen that may play a role in cardiovascular diseases. Hemagglutinins may function as adhesins and are required for virulence of several bacterial pathogens. The aim of this study was to determine the role of hemagglutinin B (HagB) in adherence of P. gingivalis to human coronary artery endothelial (HCAE) cells. P. gingivalis strain 381, a P. gingivalis 381 HagB mutant, Escherichia coli JM109 expressing HagB (E. coli-HagB), and E. coli JM109 containing pUC9 (E. coli-pUC9) were tested for their ability to attach to HCAE cells. Inhibition assays were performed to determine the ability of purified recombinant HagB (rHagB) as well as antibodies to HagB, including the polyclonal antibody (PAb) A7985 and the monoclonal antibody (MAb) HL1858, to inhibit the attachment of P. gingivalis to HCAE cells. As expected, when the attachment of P. gingivalis and the HagB mutant were compared, no statistical significance was observed between the two groups (P = 0.331), likely due to the expression of the hagB homolog hagC. However, E. coli-HagB adhered significantly better to HCAE cells than did E. coli-pUC9, the control strain. In a competition assay, the presence of purified rHagB decreased bacterial adhesion of P. gingivalis or E. coli-HagB to HCAE cells. The presence of PAb A7985 or MAb HL1858 also significantly decreased attachment of P. gingivalis and E. coli-HagB to host cells. These results indicate that HagB is involved in the adherence of P. gingivalis to human primary endothelial cells.