Danger signal-dependent activation of human dendritic cells by plasma-derived factor VIII products.

Treatment of haemophilia A by infusions of the clotting factor VIII (FVIII) results in the development of inhibitors/anti-drug antibodies in up to 25 % of patients. Mechanisms leading to immunogenicity of FVIII products are not yet fully understood. Amongst other factors, danger signals as elicited upon infection or surgery have been proposed to play a role. In the present study, we focused on effects of danger signals on maturation and activation of dendritic cells (DC) in the context of FVIII application. Human monocyte-derived DC were treated with FVIII alone, with a danger signal alone or a combination of both. By testing more than 60 different healthy donors, we show that FVIII and the bacterial danger signal lipopolysaccharide synergise in increasing DC activation, as characterised by increased expression of co-stimulatory molecules and secretion of pro-inflammatory cytokines. The degree and frequency of this synergistic activation correlate with CD86 expression levels on immature DC prior to stimulation. In our assay system, plasma-derived but not recombinant FVIII products activate human DC in a danger signal-dependent manner. Further tested danger signals, such as R848 also induced DC activation in combination with FVIII, albeit not in every tested donor. In our hands, human DC but not human B cells or macrophages could be activated by FVIII in a danger signal-dependent manner. Our results suggest that immunogenicity of FVIII is a result of multiple factors including the presence of danger, predisposition of the patient, and the choice of a FVIII product for treatment.

AP-1 Transcription Factor Serves as a Molecular Switch between Chlamydia pneumoniae Replication and Persistence.

Chlamydia pneumoniae is a Gram-negative bacterium that causes acute or chronic respiratory infections. As obligate intracellular pathogens, chlamydiae efficiently manipulate host cell processes to ensure their intracellular development. Here we focused on the interaction of chlamydiae with the host cell transcription factor activator protein 1 (AP-1) and its consequence on chlamydial development. During Chlamydia pneumoniae infection, the expression and activity of AP-1 family proteins c-Jun, c-Fos, and ATF-2 were regulated in a time- and dose-dependent manner. We observed that the c-Jun protein and its phosphorylation level significantly increased during C. pneumoniae development. Small interfering RNA knockdown of the c-Jun protein in HEp-2 cells reduced the chlamydial load, resulting in smaller inclusions and significantly lower chlamydial recovery. Furthermore, inhibition of the c-Jun-containing AP-1 complexes using tanshinone IIA changed the replicative infection phenotype into a persistent one. Tanshinone IIA-dependent persistence was characterized by smaller, aberrant inclusions, a strong decrease in the chlamydial load, and significantly reduced chlamydial recovery, as well as by the reversibility of the reduced recovery after the removal of tanshinone IIA. Interestingly, not only was tanshinone IIA treatment accompanied by a significant decrease of ATP levels, but fluorescence live cell imaging analysis by two-photon microscopy revealed that tanshinone IIA treatment also resulted in a decreased fluorescence lifetime of protein-bound NAD(P)H inside the chlamydial inclusion, indicating that chlamydial reticulate bodies have decreased metabolic activity. In all, these data demonstrate that the AP-1 transcription factor is involved in C. pneumoniae development, with tanshinone IIA treatment resulting in persistence.

Measuring the killing of intracellular pathogens: Leishmania.

Macrophages are professional phagocytes serving as a first line of defence against pathogenic organisms. Macrophages are equipped with efficient effector functions to kill invading microorganisms. The first important mechanism of macrophage host-defence is phagocytosis of pathogens. Subsequently, internalized pathogens are targeted for destruction in maturating phagolysosomal compartments. This process is mediated by lysosomal proteases and an acidified compartment. To investigate macrophages' killing potential in this chapter, we describe an assay based on human primary cells infected with the obligatory intracellular parasite Leishmania. For this pathogen the macrophage has a dual role. The parasite can use macrophages for its intracellular multiplication, but at the same time host macrophages, upon stimulation, can kill the parasite.

Apoptosis driven infection.

Professional phagocytes like polymorphonuclear neutrophil granulocytes (PMN) and macrophages (MF) kill pathogens as the first line of defense. These cells possess numerous effector mechanisms to eliminate a threat at first contact. However, several microorganisms still manage to evade phagocytic killing, survive and retain infectivity. Some pathogens have developed strategies to silently infect their preferred host phagocytes. The best example of an immune silencing phagocytosis process is the uptake of apoptotic cells. Immune responses are suppressed by the recognition of phosphatidylserine (PS) on the outer leaflet of their plasma membrane. Taking Leishmania major as a prototypic intracellular pathogen, we showed that these organisms can use the apoptotic "eat me" signal PS to silently enter PMN. PS-positive and apoptotic parasites, in an altruistic way, enable the intracellular survival of the viable parasites. Subsequently these pathogens again use PS exposition, now on infected PMN, to silently invade their definitive host cells, the MF. In this review, we will focus on L. major evasion strategies and discuss other pathogens and their use of the apoptotic "eat me" signal PS to establish infection.

Enhanced PMN response in chronic bronchitis and community-acquired pneumonia.

Chronic bronchitis is a frequent underlying disease in community-acquired pneumonia (CAP). It is unclear to what extent an impaired or exaggerated innate immune response contributes to disease manifestations and severity. To assess the role of neutrophil activation and recruitment during acute pneumonic episodes, peripheral polymorphonulcear neutrophil (PMN) activation, chemotactic activity, interleukin-8 (CXCL-8) and CXCL-8 receptor (CXCR) expression and apoptosis rate were evaluated in CAP patients with and without chronic bronchitis. In addition, the expression of CXCRs and CXCL-8 was assessed on pulmonary neutrophils in chronic bronchitis patients to compare the activation of the chemokine system in different compartments. CAP severity was assessed by the simplified acute physiology score II and the prognosis of disease was assessed by the pneumonia severity index (PSI). An increased chemotactic activity of PMN from chronic bronchitis patients with CAP was found, which was not related to the expression of CXCRs. In addition, a decreased apoptosis rate of PMN was observed. Chemotactic activity was related to the PSI. Comparison of peripheral and pulmonary PMN revealed enhanced CXCL-8 levels and a decreased CXCR expression in the lung. In conclusion, neutrophil function in patients with chronic bronchitis and community-acquired pneumonia is characterised by an increased chemotactic activity combined with a decreased apoptosis rate. The downregulation of interleukin-8 receptors in the pulmonary compartment deserves further investigation.

Phagocytes transmit Chlamydia pneumoniae from the lungs to the vasculature.

Chlamydia pneumoniae, a major cause of community-acquired pneumonia, primarily infects the respiratory tract. Chronic infection of nonrespiratory sites, such as the vascular wall, the brain or blood monocytes, requires evasion from the lungs and spreading via the bloodstream. The cell types involved in dissemination are insufficiently characterised. In this study, New Zealand White rabbits were infected intratracheally with C. pneumoniae, and lung manifestation and systemic dissemination were monitored by polymerase chain reaction and immunohistochemistry. Infection of the lungs was characterised by an early phase dominated by granulocytes and a late phase dominated by alveolar macrophages (AM). Granulocytes, AM and alveolar epithelial cells acted as host cells for chlamydiae, which remained detectable for up to 8 weeks. AM transported the pathogen to the peribronchiolar lymphatic tissue, and subsequently C. pneumoniae entered the spleen and the aorta via dissemination by peripheral blood monocytes. In conclusion, Chlamydia pneumoniae-infected alveolar macrophages transmigrate through the mucosal barrier, and give the pathogen access to the lymphatic system and the systemic circulation. Infected peripheral blood monocytes are the vector system within the bloodstream and transmit the infection to the vascular wall. This is the first description of granulocytes acting as a reservoir for Chlamydia pneumoniae early in infection.

ANCA against the bactericidal/permeability increasing protein (BPI-ANCA) can compromise the antibiotic function of BPI in a Wegener's granulomatosis patient.

A 54-year old Wegener's granulomatosis patient with PR3-ANCA at diagnosis 2 years ago was admitted with a pulmonary relapse and new subglottic stenosis preceded by pulmonary infections. The patient presented with bactericidal/permeability increasing protein (BPI)-ANCA in ELISA whereas at the same time PR3-ANCA had disappeared. Bronchoalveolar lavage revealed pulmonary infection with Gram-negative bacteria. After antibiotic treatment, immunosuppression was started with cyclophosphamide and infliximab due to refractory disease. Remission was induced and BPI-ANCA disappeared. A bacterial growth inhibition assay with BPI and the patient's IgG purified during the actual pulmonary relapse showed inhibition of the antimicrobial activity of BPI in vitro, in contrast to IgG from sera taken 2 years before and after remission was induced. The patient's BPI-ANCA recognised the bioactive N-terminal portion of BPI. Thus a possible mechanism is demonstrated for how BPI-ANCA may contribute to a pro-inflammatory setting during the development of a pulmonary relapse in the absence of PR3-ANCA by impeding bacterial clearance.

Leishmania promastigotes release a granulocyte chemotactic factor and induce interleukin-8 release but inhibit gamma interferon-inducible protein 10 production by neutrophil granulocytes.

Recent data from our laboratory suggest that neutrophil granulocytes (polymorphonuclear leukocytes [PMN]) can serve as host cells for Leishmania major in the early phase of infection. In line with these findings, an early influx of PMN to the infected tissues was shown by others to be associated with susceptibility to infection with L. major. The mechanisms underlying the initial PMN recruitment to the site of infection is poorly understood. In the present study we investigated whether Leishmania can influence PMN migration. Supernatants of Leishmania promastigotes were tested for their chemotactic activity using an in vitro chemotaxis assay. All Leishmania species tested (L. major, L. aethiopica, and L. donovani) displayed a marked chemotactic effect on human PMN. However, no effect on the migration of macrophages and NK cells was observed. Checkerboard analysis revealed that the observed PMN migration was due to chemotaxis rather than chemokinesis. Most of the chemotactic activity was found in fractions containing molecules with sizes between 10 and 50 kDa. Pretreatment of PMN with N-formyl-methionyl-leucyl-phenylalanine blocked the chemotactic activity of Leishmania supernatants up to 75%. In addition, we found that leishmanial contact induced the release of interleukin-8 (IL-8) and inhibited the production of gamma interferon-inducible protein 10 (IP-10) by PMN. These data suggest that infection with Leishmania promastigotes leads to PMN accumulation via the production of a chemotactic factor by the parasites, and this effect is amplified by the induction of IL-8 production in PMN. On the other hand, the inhibition of IP-10 production can lead to prevention of NK cell activation.

Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes.

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.

Chemokines, natural killer cells and granulocytes in the early course of Leishmania major infection in mice.

In the present study the early recruitment of leukocytes into the infected skin and into the draining lymph node (LN) was investigated after subcutaneous infection of mice with Leishmania major promastigotes. Flow cytometric analysis of cells recovered from the infected skin revealed that GR-1+ granulocytes were present as early as 10 h after infection, thus representing the first leukocyte population to be recruited to the site of infection. The migration of granulocytes was shown to be associated with a rapid mRNA expression of the neutrophil-attracting chemokine KC in the infected skin. Moreover, L. major promastigotes were found to produce factor(s) that are chemotactic for human neutrophils in vitro. Experiments with human neutrophils revealed that these cells are able to phagocytose the parasites. Natural killer (NK) cells appeared at the site of infection 24 h after infection. The migration of NK cells in resistant mice was found to correlate with the expression of the NK cell-activating chemokine IP-10. Treatment of susceptible BALB/c mice with recombinant mouse IP-10 resulted in a significantly increased NK cell cytotoxic activity in the draining LN. These data suggest that both the early chemokine gene expression and the production of chemotactic factors by the parasites themselves regulate the site-directed migration and activation of cells of the innate immune response, and suggest a role of chemotactic factors in the early defense against the parasites.

FcalphaRI-positive liver Kupffer cells: reappraisal of the function of immunoglobulin A in immunity.

Despite the well-recognized involvement of immunoglobulin (Ig) A in mucosal immunity, the function of its receptor, FcalphaRI (CD89), is poorly understood. The ability of FcalphaRI to activate leukocytes seems to conflict with the proposed anti-inflammatory activity of secretory IgA. We show here that in a transgenic mouse model, inflammatory mediators induced expression of FcalphaRI on Kupffer cells, which enabled efficient phagocytosis in vivo of bacteria coated with serum IgA. Secretory IgA did not initiate phagocytosis. Therefore, interactions between serum IgA and FcalphaRI on Kupffer cells may provide a 'second line of defense' in mucosal immunity, by eliminating invasive bacteria entering through the portal circulation and thus preventing disease.

Crosslinking of the human Fc receptor for IgA (FcalphaRI/CD89) triggers FcR gamma-chain-dependent shedding of soluble CD89.

CD89/FcalphaRI is a 55- to 75-kDa type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. At present, no information is available on the existence of soluble forms of this receptor. We developed an ELISA for the detection of soluble CD89 (sCD89) forms and investigated the regulation of sCD89 production. PMA/ionomycin stimulation of monocytic cell lines (U937, THP-1, and MM6), but not of neutrophils, resulted in release of sCD89. Crosslinking of CD89 either via its ligand IgA or with anti-CD89 mAbs similarly resulted in sCD89 release. Using CD89-transfected cells, we showed ligand-induced shedding to be dependent on coexpression of the FcR gamma-chain subunit. Shedding of sCD89 was dependent on signaling via the gamma-chain and prevented by addition of inhibitors of protein kinase C (staurosporine) or protein tyrosine kinases (genistein). Western blotting revealed sCD89 to have an apparent molecular mass of 30 kDa and to bind IgA in a dose-dependent fashion. In conclusion, the present data document a ligand-binding soluble form of CD89 that is released upon activation of CD89-expressing cells. Shedding of CD89 may play a role in fine-tuning CD89 immune effector functions.

Immunoglobulin-binding sites of human FcalphaRI (CD89) and bovine Fcgamma2R are located in their membrane-distal extracellular domains.

To localize the immunoglobulin (Ig)-binding regions of the human Fcalpha receptor (FcalphaRI, CD89) and the bovine Fcgamma2 receptor (bFcgamma2R), chimeric receptors were generated by exchanging comparable regions between these two proteins. FcalphaRI and bFcgamma2R are highly homologous and are more closely related to each other than to other human and bovine FcRs. Nevertheless, they are functionally distinct in that FcalphaRI binds human IgA (hIgA) but not bovine IgG2 (bIgG2), whereas bFcgamma2R binds bIgG2 but not hIgA. FcalphaRI and bFcgamma2R possess extracellular regions consisting of two Ig-like domains, a membrane-distal extracellular domain (EC1), a membrane-proximal EC domain (EC2), a transmembrane region, and a short cytoplasmic tail. Chimeras constructed by exchanging complete domains between these two receptors were transfected to COS-1 cells and assayed for their ability to bind hIgA- or bIgG2-coated beads. The results showed that the Ig-binding site of both FcalphaRI and bFcgamma2R is located within EC1. Supporting this observation, monoclonal antibodies that blocked IgA binding to FcalphaRI were found to recognize epitopes located in this domain. In terms of FcR-Ig interactions characterized thus far, this location is unique and surprising because it has been shown previously that leukocyte FcgammaRs and FcepsilonRI bind Ig via sites principally located in their EC2 domains.

Human mesangial cells in culture and in kidney sections fail to express Fc alpha receptor (CD89).

The mechanism of deposition of IgA in the renal mesangium in primary IgA-nephropathy is poorly understood. It has been suggested that membrane receptors for IgA on mesangial cells (MC) of the kidney may be involved. To obtain more insight in the occurrence of the myeloid receptor for IgA (CD89) on MC, both in situ and in culture, rabbit and goat polyclonal antibodies and mouse monoclonal antibody against recombinant CD89 were raised. Kidney sections from five control subjects and five patients with primary IgA-nephropathy failed to be positive for CD89 in the mesangium, using our polyclonal and monoclonal antibodies. Also, five primary human MC cultures assessed for CD89 expression showed no protein expression of CD89. Furthermore, reverse transcription-PCR failed to detect mRNA expression of CD89 in the cultured MC. It was demonstrated that all five human primary MC bound human IgA in a dose-dependent manner, which was not inhibitable by blocking monoclonal anti-CD89 antibody (My43). In contrast, binding of IgA to U937 cells was blocked efficiently by My43. Finally, incubation of human MC with either human or rat IgA led to increased interleukin-6 production, whereas only human IgA, but not rat IgA, was able to bind to human CD89. Therefore, it is concluded that human MC do not express CD89 (to a significant extent). These results strongly suggest that binding of IgA to human MC occurs via an IgA receptor distinct from CD89.

Reduced binding of immunoglobulin A (IgA) from patients with primary IgA nephropathy to the myeloid IgA Fc-receptor, CD89.

BACKGROUND: Primary IgA nephropathy (IgAN) is associated with elevated levels of circulating IgA and is characterized by deposition of primarily IgA1 in the renal mesangium. It has not yet been clarified which mechanisms govern the deposition of IgA1 in the mesangium. One of the factors which may play a role in trapping of IgA in the mesangial area is the interaction of IgA with specific IgA receptors (Fc alphaR, CD89) on the mesangial cells. METHODS: In the present study IgA derived from patients with IgAN and controls was investigated for its interaction with human CD89, expressed on the surface of the murine B cell line IIA1.6. RESULTS: IgA binding to CD89 expressing cells was specific, concentration dependent and binding of dIgA and pIgA occurred in a more efficient fashion than that of mIgA. IgA binding to CD89 directly from serum of patients compared to controls showed no significant difference. However these experiments are affected by differences in IgA concentration and combinations of different sizes of IgA. Using purified fractions of mIgA, dIgA, and pIgA isolated from serum, a significantly reduced binding of mIgA to CD89 from patients compared to controls was observed. Finally, the binding of aIgA2 to CD89 was less inhibited using mIgA from patients with IgAN compared to controls. CONCLUSIONS: The reduced binding of mIgA to CD89 seems to contradict a direct role for CD89 in deposition of IgA. However reduced binding of mIgA to CD89 may affect IgA clearance, leading to higher serum IgA. Furthermore, since it has been demonstrated that mIgA can interfere with binding of di- and pIgA, CD89 could still contribute to pIgA deposition in the mesangial area.

Size-dependent effect of IgA on the IgA Fc receptor (CD89).

The IgA Fc receptor (FcR; CD89) is expressed on several types of cells of the myeloid cell lineage. We investigated whether different sizes of heat-aggregated IgA (aIgA) bind to CD89 and subsequently induce cellular activation. As a model we used the murine B cell line IIA1.6 transfected with CD89 or IIA1.6 cells transfected with CD89 as well as with the FcR gamma chain to study the binding of IgA to CD89. When these cells expressing CD89 were incubated with monomeric IgA, no significant binding of IgA to the cells was detectable by fluorescence-activated cell sorter analysis; however, incubation of the cells with aggregated IgA resulted in 93 +/- 2% positive cells. Incubation of the cells with different sizes of IgA-containing aggregates revealed optimal binding with aggregates containing five to six molecules of IgA per aggregate. No difference was observed between the binding to CD89 of both IgA1- or IgA2-containing aggregates. Furthermore, the binding of aIgA was found to be CD89-specific, since the binding of IgA was completely inhibited by the CD89-specific monoclonal antibody My43 and no detectable binding occurred to the IIA1.6 parent cell line. Activation studies using interleukin-2 (IL-2) production as a marker, showed that the FcR gamma chain is necessary to induce cellular activation. Only cells transfected with both CD89 and the FcR gamma chain (CD89+/gamma +) enhance the IL-2 production 10-12-fold upon stimulation with aggregates of IgA. Furthermore, triggering of CD89 only results in increase of intracellular calcium concentration ([Ca2+]i) in cells co-expressing FcR gamma chain. Mutation of the tyrosine residues in the FcR gamma chain immunoreceptor tyrosine-based activation motif of the FcR gamma chain abolishes this increase in [Ca2+]i, indicating association and involvement of the FcR gamma chain in CD89-mediated signaling.