Gertrud Maria Hänsch

Polymorphonuclear neutrophils and T lymphocytes: strange bedfellows or brothers in arms?

Polymorphonuclear neutrophils (PMN) are linked invariably to the innate immune response, particularly to the defence against bacterial infection. T lymphocytes are studied mainly in virus infections, the defence against tumours, the development and progression of chronic inflammatory processes, in autoimmune phenomena and in materno-fetal tolerance. There is, however, increasing evidence for communication and interactions between PMN and T cells that we discuss here in the context of different physiological and pathological conditions, including acute and chronic inflammatory disease, defence against tumours, and maintenance of pregnancy.

MCP-1 Induces Inflammatory Activation of Human Tubular Epithelial Cells: Involvement of the Transcription Factors, Nuclear Factor-κB and Activating Protein-1

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine synthesized by several cell types, e.g., inflammatory cells, such as monocytes, and resident renal cells, such as human tubular epithelial cells (TECs). Besides induction of monocyte recruitment, MCP-1 has been suggested to induce non-leukocytes to produce cytokines and adhesion molecules. Inflammation of the tubulointerstitium is a hallmark of many renal diseases and contributes to progression of renal failure; the purpose therefore of this study was to investigate the influence of MCP-1 on markers of inflammatory activation in human TECs. MCP-1 stimulated interleukin-6 (IL-6) secretion and intercellular adhesion molecule-1 (ICAM-1) synthesis in a time- and dose-dependent manner. In parallel, MCP-1 increased IL-6 and ICAM-1 mRNA expression in human TECs. Pretreatment with pertussis toxin, GF109203X, BAPTA-AM, and pyrrolidine dithiocarbamate inhibited MCP-1-dependent IL-6 and ICAM-1 synthesis, suggesting the involvement of Gi-proteins, protein kinase C, intracellular Ca(2+), and nuclear factor-kappaB (NF-kappaB) in MCP-1 signaling. Using electrophoretic gel mobility shift assay, we observed that MCP-1 stimulated binding activity of NF-kappaB. Binding activity of the activator protein-1 (AP-1), which has been implicated to regulate induction of the IL-6 gene together with NF-kappaB, was also stimulated by MCP-1. In the present experiments, NF-kappaB and AP-1 were involved in the MCP-1-mediated induction of IL-6, as demonstrated by cis element double-stranded (decoy) oligonucleotides (ODN). In contrast to IL-6 release, MCP-1-induced ICAM-1 expression was predominantly dependent on NF-kappaB activation. These results document for the first time that MCP-1 induces an inflammatory response in human TECs. This may be an important new mechanism in the pathogenesis of tubulointerstitial inflammation.

Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation

Polymophonuclear cells (PMN) of healthy donors do not express major histocompatibility complex (MHC) class II antigens or the T‐cell costimulatory molecules CD80 or CD86. Expression of these receptors, however, is seen in patients with chronic inflammatory diseases. We now report that, by culturing PMN of healthy donors with autologous serum, interferon‐γ (IFN‐γ) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF), de novo synthesis of MHC class II, CD80 and CD86 could be induced. MHC class II‐positive PMN acquired the capacity to present staphylococcus enterotoxin to peripheral T cells, apparent as induction of interleukin‐2 (IL‐2) synthesis and proliferation of the T cells. Moreover, the PMN also processed tetanus toxoid (TT) and induced proliferation of TT‐specific T cells in a MHC class II‐restricted manner. Taken together, these data indicate that PMN can be activated to function as accessory cells for T‐cell activation.

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.

Effect of the Late Complement Components C5b-9 on Human Monocytes: Release of Prostanoids, Oxygen Radicals and of a Factor Inducing Cell Proliferation

Recently, we reported stimulation of rat macrophages and human platelets by isolated C5b-9 to synthesize prostaglandin E (PGE) or thromboxane B2 (TXB2). In the present study, we tested whether besides prostanoids, C5b-9 also would induce the production of other mediators. We found that C5b-9 in sublytic concentrations stimulated human granulocytes (polymorphonuclear leukocytes) or monocytes to release oxygen radicals. Furthermore, monocytes release interleukin-1 in response to C5b-9. Thus, besides having a lytic capacity, C5b-9 also functions as a stimulator of various cells.

Oxygen Radical Generation in Human Platelets: Dependence on 12-Lipoxygenase Activity and on the Glutathione Cycle

Stimulation of the arachidonic acid metabolism in human platelets also induces chemiluminescence, which indicates the formation of highly reactive oxygen species. To analyze the pathway by which oxygen radicals are generated, we tested the dependence of O2- generation on the eicosanoid metabolism. We found that inhibition of lipoxygenase, but not of cyclooxygenase, prevented the O2- production. Moreover, depletion of cellular glutathione peroxidase resulted in reduced synthesis of the lipoxygenase product 12-hydroxyeicosatetraenoic acid, as well as in reduce O2- thus establishing a link between O2- generation, the lipoxygenase pathway and the glutathione cycle.

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.

Systemic Staphylococcus aureus infection mediated by Candida albicans hyphal invasion of mucosal tissue

Candida albicans and Staphylococcus aureus are often co-isolated in cases of biofilm-associated infections. C. albicans can cause systemic disease through morphological switch from the rounded yeast to the invasive hyphal form. Alternatively, systemic S. aureus infections arise from seeding through breaks in host epithelial layers although many patients have no documented portal of entry. We describe a novel strategy by which S. aureus is able to invade host tissue and disseminate via adherence to the invasive hyphal elements of Candida albicans. In vitro and ex vivo findings demonstrate a specific binding of the staphylococci to the candida hyphal elements. The C. albicans cell wall adhesin Als3p binds to multiple staphylococcal adhesins. Furthermore, Als3p is required for C. albicans to transport S. aureus into the tissue and cause a disseminated infection in an oral co-colonization model. These findings suggest that C. albicans can facilitate the invasion of S. aureus across mucosal barriers, leading to systemic infection in co-colonized patients.

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.