Christof Wagner

The complement receptor 3, CR3 (CD11b/CD18), on T lymphocytes: activation-dependent up-regulation and regulatory function.

The complement receptor 3 (CR3; CD11b/CD18) is present exclusively on leukocytes, particularly on NK cells, monocytes and polymorphonuclear neutrophils. Approximately 10% of peripheral T lymphocytes and, as we found now mainly CD8+ cells, expressed CD11b. Upon stimulation, however, expression of CD11b was up‐regulated also on CD4+ cells. Stimulation of T cells either bycross‐linked anti‐CD3 and IL‐2 or by mononuclear cells and mitogen yielded up to 28% CD11b+ T cells. The majority of CD11b+ T cells also expressed CD56. T cell lines established from healthy donors were also found to express CR3. When restimulated up to 90% of cells became positive for CD11b making those cells an ideal tool for studying the functional role of CD11b. Antibodies to CD11b and bona fide ligands for the complement receptor inhibited the anti‐CD3‐induced T cell proliferation and as well as IL‐2 release . In contrast, proliferation of a CD11b– T cell line was not inhibited. Taken together, our data indicate an activation‐dependent expression of the complement receptor on T cells and suggest a regulatory function.

Induction of Neutrophil Chemotaxis by the Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

ABSTRACT Acyl homoserine lactones are synthesized by Pseudomonas aeruginosa as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.

Transdifferentiation of polymorphonuclear neutrophils : acquisition of CD83 and other functional characteristics of dendritic cells

Polymorphonuclear neutrophils (PMN) are in the first line of defense against bacterial infections. They are considered to be end-differentiated cells undergoing constitutive apoptosis within hours after release from the bone marrow. During pathological events, however, their life span is extended in conjunction with morphological and functional alterations indicative of a transdifferentiation of mature PMN. To further characterize differentiated PMN, the alterations seen in vivo were reproduced by cultivating PMN of healthy donors with either γ-interferon, granulocyte/macrophage colony stimulating factor, or a combination thereof. Thus cultivated cells escaped from apoptosis, and protein synthesis was induced, notably of the major histocompatibility complex (MHC) class II antigens, CD80 and CD86. Moreover, CD83, thought to be specific for dendritic cells was synthesized, while typical markers of PMN, including CD66b, CD11a/CD11b/CD11c, CD15, CD18 were preserved. A profound alteration of both cellular morphology and of function was seen: the cultivated PMN lost their chemotactic activity but had acquired the ability to present to T-cells a peptide antigen in a MHC class II restricted manner. The data lead to the conclusion that mature PMN can differentiate further to cells with characteristics of DCs, thereby connecting PMN to the specific T-cell response.

Up-regulation of the dendritic cell marker CD83 on polymorphonuclear neutrophils (PMN): divergent expression in acute bacterial infections and chronic inflammatory disease

Upon cultivation with interferon‐γ (IFN‐γ ) and granulocyte/macrophage‐colony stimulating factor (GM‐CSF) polymorphonuclear neutrophils (PMN) acquire characteristics of dendritic cells, including expression of major histocompatibility complex (MHC) class II antigens, of the co‐stimulatory antigens CD80, CD86 and of CD83, the latter considered to be specific for dendritic cells. Dendritic‐like PMN were also able to present to T cells antigens in a MHC class II‐restricted manner. To assess whether dendritic‐like PMN are also generated in vivo, cells of patients with acute bacterial infections and of patients with chronic inflammatory diseases (primary vasculitis) were tested. During acute infection up to 80% of PMN acquired CD83, but remained negative for MHC class II, CD80 or CD86. PMN of patients with primary vasculitis expressed MHC class II antigens, CD80 and CD86, but not CD83, indicating that up‐regulation of MHC class II and of CD83 are not necessarily linked to each other. Indeed, parallel studies with PMN of healthy donors showed that while IFN‐γ and granulocyte/macrophage colony stimulating factor (GM‐CSF) induced both, MHC class II and CD83, tumour necrosis factor (TNF)‐α selectively induced de novo synthesis of CD83. The function of CD83 on PMN is still elusive. A participation in the MHC class II‐restricted antigen presentation could be ruled out, consistent with the segregation of MHC class II and CD83 expression. Regardless, however, of its function, CD83 expression could serve as a marker to differentiate between acute and chronic inflammation.

Lipopolysaccharides (LPS) induce the differentiation of human monocytes to osteoclasts in a tumour necrosis factor (TNF) alpha-dependent manner: a link between infection and pathological bone resorption.

The degradation of bone is a serious consequence of persistent bacterial infection, including periodontitis, infection-associated non-unions or osteomyelitis. To test the hypothesis that infection and inflammatory conditions promote the differentiation of monocytes to bone-resorbing osteoclasts, highly purified monocytes, or alternatively, cells of the promyeloid cell line U937, differentiated to monocyte-like cells, were cultivated in the presence of lipopolysaccharides (LPS) for up to 30 days. After 2-4 days, a massive aggregation of the cells was observed, after 15-20 days multinuclear cells with the morphological characteristics of osteoclasts became apparent. These cells expressed the osteoclast-typical proteins tartrate-resistant acid phosphate (TRAcP) and cathepsin K. Moreover, these cells formed resorption pits on calcium phosphate coated cover slips or ivory slices. To test whether the differentiation of the monocytes to osteoclast-like cells was mediated by tumour necrosis factor alpha (TNFalpha) secreted by the cells in culture, an antibody directed against TNFalpha was added together with LPS. Differentiation to osteoclast-like cells was inhibited, suggesting a paracrine effect of locally produced TNFalpha. In conclusion, we propose that local bacterial infections could create a microenvironment that promotes the generation of bone resorbing cells, which, in turn, could contribute to the infection-associated osteolysis.

Expression patterns of the lipopolysaccharide receptor CD14, and the FCγ receptors CD16 and CD64 on polymorphonuclear neutrophils: Data from patients with severe bacterial infections and lipopolysaccharide-exposed cells

In polymorphonuclear neutrophils (PMN) CD14, one of the receptors for lipopolysaccharides (LPS) is stored intracellularly as a preformed protein, with only few receptors expressed on the surface. We now report that in patients with severe bacterial infections, CD14 expression is profoundly upregulated, as is CD64 (Fc&ggr;RI), the high-affinity receptor for IgG, whereas CD16 (Fc&ggr;RIII) was partly lost from the surface. To further analyze regulation of these receptors, PMN of healthy donors were exposed to low doses of LPS. By brief exposure (10-120 min) to LPS, CD14 was transferred to the surface in a cytochalasin B-sensitive manner, as were CD16 and CD64. Prolonged culture (up to 48 h) resulted in a further upregulation of CD14, sustained expression of CD64, and profound decline of CD16, yielding a similar pattern of receptor expression as seen in the patients. Subsequent studies revealed that LPS induced de novo synthesis of CD14: the increase of surface expression could be inhibited by cycloheximide and by interfering with a known LPS-induced signaling event, the translocation of NF&kgr;B. Moreover, an up to 10-fold increase of specific mRNA was seen, as was incorporation into CD14 of 35S-methionine. The de novo synthesis prolonged expression of CD14, whereas the CD16 expression declined, generating a PMN phenotype characteristic for severe infection and indicative of escape from apoptosis of a PMN subpopulation.

Cellular inflammatory response to persistent localized Staphylococcus aureus infection: phenotypical and functional characterization of polymorphonuclear neutrophils (PMN)

Persistent, localized Staphylococcus aureus infections, refractory to antibiotic treatment, can result in massive tissue destruction and surgical intervention is often the only therapeutic option. In that context, we investigated patients with S. aureus‐induced infection at various sites, apparent as either olecranon bursitis, empyema of the knee joint or soft tissue abscess formation. As expected, a prominent leucocyte infiltrate was found, consisting predominantly of polymorphonuclear neutrophils (PMN) (up to 75%) and to a lesser extent of T lymphocytes and natural killer (NK) cells. In line with their bactericidal capacity, PMN expressed the high‐affinity receptor for IgG, CD64 and the lipopolysaccharide (LPS) receptor CD14; moreover, the oxygen radical production in response to the bacterial peptide f‐MLP was enhanced, while chemotactic activity was greatly reduced. The more intriguing finding, however, was that a portion of PMN had acquired major histocompatibility complex (MHC) class II antigens and CD83, indicative of a transdifferentiation of PMN to cells with dendritic‐like characteristics. Of note is that a similar transdifferentiation can be induced in PMN in vitro, e.g. by gamma interferon or by tumour necrosis factor alpha. Co‐cultivation of transdifferentiated PMN with autologous T lymphocytes resulted in prominent T cell proliferation, provided that S. aureus enterotoxin A was added. Taken together, persistent S. aureus infection induces PMN to acquire characteristics of dendritic cells, which in turn might promote the local immune response.

Treatment of tibial shaft non-unions: bone morphogenetic proteins versus autologous bone graft

Fractures of the tibial shaft are likely to result in delayed union or non-union; 10-30% of these fractures will not heal properly and are commonly treated with autologous bone grafting. BMP-7 is a recombinant bone growth factor that can be applied locally as an alternative or in addition to autologous bone grafting, and this study aimed to compare the efficiency of the two procedures. From January 1995 to December 2002, 82 people with delayed union of a tibial fracture at least 4 months after primary stabilisation underwent autologous bone grafting. Successful healing was defined as radiological bony consolidation. Between May 2002 and June 2005, 26 similar cases were treated after the failure of the graft with local implantation of BMP-7, and were followed up for at least 1 year. Of the 82 people receiving autologous bone grafts only, 24 (28%) still had no signs of consolidation after 4 months and required revision surgery. Of the 26 people with BMP-7 implantation after failed graft, bony consolidation was seen after 4 months in 24 cases and only 2 (8%) required revision surgery. The BMP-7 group, although including more complicated cases, showed a significantly higher (p = 0.025) success rate compared with the group that did not receive BMP-7.

Polymorphonuclear neutrophils in posttraumatic osteomyelitis: cells recovered from the inflamed site lack chemotactic activity but generate superoxides.

The pathogenesis of posttraumatic osteomyelitis, one of the major complications after orthopedic surgery, is not yet understood. Formation of bacterial biofilms on the implant is presumed, conferring resistance to antibiotic therapy and probably also to the host defense mechanisms. In that context, the polymorphonuclear neutrophils (PMN) having infiltrated the infected site were recovered and characterized phenotypically and functionally. Loss of CD62L and upregulation of CD14 were seen, as was expression of CD83. Expression of the latter is dependent on de novo protein synthesis and thus is indicative of an extended life span and a transdifferentiation of the PMN at the infected site. The infiltrated PMN had lost their chemotactic activity, whereas the capacity to produce superoxides was preserved and in some patients even enhanced. In vitro experiments done in parallel showed that long-term culture with interferon-γ resulted in similar alterations of PMN: loss of chemotactic activity, whereas other functions of PMN, such generation of superoxides and phagocytosis of opsonized bacteria, were preserved or even enhanced. The loss of the migratory capacity of PMN having already emigrated from the blood vessel to the infected site is not expected to affect the host defense negatively. Assuming, however, that bacteria are organized as a biofilm and that infiltration into this biofilm is required for phagocytosis of the bacteria, our data could to some extent explain why despite being activated, the PMN are not able to control the infection. By releasing their cytotoxic, proteolytic, and collagenolytic potential, PMN might instead contribute to tissue destruction and eventually to osteolysis.

Biofilm growth on implants: bacteria prefer plasma coats.

Purpose Bacterial biofilm formation on prostheses or devices used for osteosynthesis is increasingly recognized as cause of persistent infections, an entity known as implant-associated posttraumatic osteomyelitis. Biofilm formation is a very complex, multistep process with adhesion as the first and decisive step. The most prevalent pathogens found are staphylococci species, especially S. aureus, presumably due to a preference to non-biological materials, such as metal. Adherence is influenced by several factors, including the microenvironment, in which blood proteins from serum or plasma might influence adhesion and maybe biofilm formation. The aim of the present study was to test and to compare adherence of S. aureus and P. aeruginosa to different biological and non-biological surfaces in vitro. The question was addressed if coating of the surface by plasma or serum proteins influences bacterial adherence. Methods Adherence of radiolabeled bacteria to different surfaces in the presence or absence or serum/plasma proteins was measured over time. Results When testing adherence of S. aureus to plastic, titanium or to monolayers of epithelial cells (A549) or fibroblasts (Colo800) a clear-cut preference for non-biological surfaces, especially for titanium was seen. Using P. aeruginosa species a similar pattern without a significant difference was revealed. When mimicking the in vivo situation by pre-coating of titanium with human serum or plasma adherence was increased, especially when titanium was coated (“opsonized”) by plasma. Conclusions Bacterial adherence to surfaces is determined by a variety of factors such as temperature, the presence of nutrients, the absence of host defense systems and the configuration of the covered surface. In vivo, adherence to non-biological surfaces is also influenced by the microenvironment, especially plasma proteins, promoting biofilm formation.