Molecular view on PRR cross-talk in antifungal immunity.

The identification of a major class of innate immune receptors, termed pattern recognition receptors (PRRs), has boosted research on innate pathogen recognition. The immune response to a specific pathogen is not restricted to the recognition by one type of PRR or activation of a single cell type, but instead comprises complex collaborations between different receptors, cells and signal mediators. Here we will discuss the cross-talk between PRRs involved in fungal recognition, focusing on the molecular interactions occurring at the plasma membrane.

Tetraspanin microdomains in immune cell signalling and malignant disease.

A contemporary goal of researchers in leucocyte signalling has been to uncover how cells physically organize and compartmentalize signalling molecules into efficient, regulated signalling networks. This work has revealed important roles of membrane microdomains that are characterized by their distinctive protein and lipid compositions. Recent studies have demonstrated that besides typical cholesterol- and glycosphingolipid-enriched 'rafts', leucocyte membranes are equipped with a different type of microdomain, made up of tetraspanin proteins. Tetraspanin proteins are involved in the organization of tetraspanin-enriched microdomains by virtue of their capacity to specifically associate with key molecules, including integrins, leucocyte receptors and signalling proteins. The aspects of leucocyte function influenced by tetraspanin microdomains include adhesion, proliferation and antigen presentation. However, the mechanisms by which tetraspanin complexes link to intracellular signalling pathways, are still largely unknown. This review discusses how tetraspanin microdomains might function to regulate signalling in lymphoid and myeloid cells, and how they relate to lipid rafts. In addition, we discuss new insights into the role of tetraspanins in malignant disease.

Novel immunotherapeutic strategies for invasive fungal disease.

Infectious diseases still represent a major cause of mortality in man. Failure of the immune system to eradicate pathogens may result in serious invasive infections. Increasing numbers of immunocompromised patients, ineffective treatment and the emergence of drug-resistant pathogens represent the underlying problems. The focus of this review is on the pathogenesis of systemic fungal disease and the development of novel immunotherapeutic approaches. A comprehensive understanding of host-pathogen interactions is required for manipulation of immune responses to direct anti-fungal immunity. The potential of growth factor- and antibody-based therapeutics for invasive fungal disease are discussed. Recent advances in antibody technology may provide new strategies for developing antibody-based anti-fungal therapies. Development of fungal disease is related primarily to impaired host defense mechanisms, which emphasizes the importance of immunorestorative therapy in immunocompromised individuals. Controlled comparative clinical research is needed to evaluate the efficacy of novel immunotherapies, which provide prospects for the future management of invasive fungal disease.

Immunoglobulin A-mediated protection against Bordetella pertussis infection.

Infection with Bordetella pertussis, the causative agent of pertussis (whooping cough) in humans, is followed by the production of antibodies of several isotypes, including immunoglobulin A (IgA). Little is known, however, about the role of IgA in immunity against pertussis. Therefore, we studied targeting of B. pertussis to the myeloid receptor for IgA, FcalphaRI (CD89), using either IgA purified from immune sera of pertussis patients or bispecific antibodies directed against B. pertussis and FcalphaRI (CD89 BsAb). Both IgA and CD89 BsAb facilitated FcalphaRI-mediated binding, phagocytosis, and bacterial killing by human polymorphonuclear leukocytes (PMNL) and PMNL originating from human FcalphaRI-transgenic mice. Importantly, FcalphaRI targeting resulted in enhanced bacterial clearance in lungs of transgenic mice. These data support the capacity of IgA to induce anti-B. pertussis effector functions via the myeloid IgA receptor, FcalphaRI. Increasing the amount of IgA antibodies induced by pertussis vaccines may result in higher vaccine efficacy.

Neutrophil Fc gamma RI as target for immunotherapy of invasive candidiasis.

Invasive candidiasis represents a life-threatening disease for immunocompromised patients. This study focused on new immunotherapeutic approaches for systemic Candida albicans infections in a human FcgammaRI-transgenic mouse model. FcgammaRI (CD64) is a potent immunoactivating receptor on phagocytic and dendritic cells. In vivo targeting of C. albicans toward neutrophil-FcgammaRI by bispecific Abs and G-CSF effectively protected FcgammaRI-transgenic mice from lethal candidiasis. Nontransgenic mice were not protected, and treatment with bispecific Ab or G-CSF alone did not reduce mortality. Furthermore, infected FcgammaRI-transgenic mice developed high titers of anti-C. albicans IgG, and survival was extended on secondary infection without further treatment. These findings document the capacity of FcgammaRI to initiate potent anti-C. albicans immunity and support the development of FcgammaRI-directed immunotherapy of invasive fungal disease.

Enhancement of polymorphonuclear cell-mediated tumor cell killing on simultaneous engagement of fcgammaRI (CD64) and fcalphaRI (CD89).

Antibodies can efficiently induce antitumor responses via recruitment of Fc receptor-bearing cytotoxic cells. Polymorphonuclear (PMN) cells represent attractive effector cells for antibody-directed immunotherapy. This, because activated PMN cells coexpress the class I receptors for IgG (FcgammaRI, CD64) and IgA (FcalphaRI, CD89), which are potent cytotoxic trigger molecules. Both receptors, however, require the FcR gamma chain for signaling. In this study, we show that FcgammaRI and FcalphaRI can trigger function independently of one another and do not cross-compete for the FcR gamma chain. FcalphaRI proved more efficient in initiating early signaling events and effector functions, such as redirected tumor cell killing and generation of superoxide. In addition, simultaneous engagement of FcgammaRI and FcalphaRI resulted in enhanced tumor cell lysis. These data support the development of concepts in which both FcgammaRI and FcalphaRI on PMN cells are targeted for tumor therapy.

Effective in vitro clearance of Porphyromonas gingivalis by Fc alpha receptor I (CD89) on gingival crevicular neutrophils.

Porphyromonas gingivalis has been implicated as a causative pathogen in periodontitis. Immunotherapeutic approaches have recently been suggested to aid in the clearance of P. gingivalis from disease sites. Because antibody-Fc receptor (FcR) interactions play a role in the effector functions of polymorphonuclear neutrophils (PMN), we evaluated which FcR on PMN from gingival crevicular fluid (GCF) serves as an optimal target molecule for FcR-directed immunotherapy. GCF PMN and peripheral blood (PB) PMN from adult periodontitis patients were analyzed for their immunoglobulin G (IgG) and IgA FcR (Fc gamma R and Fc alpha R, respectively) expression and function by studying IgG- and IgA-mediated elimination of P. gingivalis. GCF PMN exhibited higher Fc alpha RI and Fc gamma RI levels and lower Fc gamma RIIa and Fc gamma RIIIb levels than PB PMN. Functional studies revealed that GCF PMN exhibited less of a capacity to phagocytose and kill IgG1-opsonized P. gingivalis than PB PMN. IgA1-mediated phagocytosis and killing capacity was, however, comparable between GCF PMN and PB PMN. In summary, these in vitro results document that Fc alpha RI represents a candidate target for FcR-directed immunotherapy for the clearance of P. gingivalis.

Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation.

Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.

IgA and the IgA Fc receptor.

IgA has traditionally been regarded a non-inflammatory antibody. This might indeed be true for secretory IgA (SIgA), which exerts its function at mucosal surfaces where commensal microorganisms and dietary antigens prevail. Serum IgA, however, potently triggers (pro)-inflammatory activity upon binding to the myeloid IgA receptor, FcalphaRI. Here, new insights in the roles of IgA and FcalphaRI are addressed and a model integrating the various functions of IgA in immunity is discussed.

Targeting to Fcgamma receptors, but not CR3 (CD11b/CD18), increases clearance of Bordetella pertussis.

In the absence of opsonizing antibodies, Bordetella pertussis, the causative agent of pertussis, readily binds to phagocytes via complement receptor 3 (CR3). After opsonization with antibodies, binding is mediated by IgG receptors (FcgammaR). The effect of targeting B. pertussis to either FcgammaR or CR3 was studied. The fate of unopsonized B. pertussis, IgG-opsonized B. pertussis, and B. pertussis opsonized with bispecific antibodies (BsAbs) directed to CR3 or FcgammaRII/-III was compared. IgG antibodies mediated binding and phagocytosis of B. pertussis via FcgammaR by polymorphonuclear leukocytes (PMNL) in vitro. Opsonization of B. pertussis with BsAbs directed against either CR3 or FcgammaRII/-III facilitated PMNL phagocytosis; however, in vivo studies with BsAb revealed that FcgammaR-mediated uptake facilitates B. pertussis clearance, in contrast to uptake via CR3. Targeting of B. pertussis to FcgammaRII/-III in mice deficient in FcgammaRII or FcgammaRIII indicated that the protective effect is attributable to FcgammaRIII. Competition between uptake via CR3 or FcgammaR may determine the outcome of natural infection.

Role of pulmonary surfactant protein D in innate defense against Candida albicans.

Pulmonary surfactant protein D (SP-D), which is a member of the collectin family, is implicated in pulmonary defense against pathogens. To determine whether SP-D is involved in first-line immunity against Candida albicans, an important respiratory fungus, the interaction of SP-D with C. albicans was studied. SP-D was found to bind C. albicans, resulting in agglutination of the microorganisms. Binding was calcium dependent and was inhibited by competing sugars maltose or mannose. Incubation of C. albicans with SP-D resulted in profound fungal growth inhibition and decreased hyphal outgrowth. Furthermore, it was found that SP-D inhibited phagocytosis of C. albicans by alveolar macrophages. These data suggest that the lung collectin SP-D has an important role in first-line defense against C. albicans in the lung, by agglutinating C. albicans and limiting their growth, without the need for macrophage activation.

Immunotherapeutic perspective for bispecific antibodies.

Bispecific antibodies (BsAb) can, by virtue of combining two binding specificities, improve the selectivity and efficacy of antibody-based treatment of human disease. Recent studies underline the importance of both the 'anti-trigger' and 'anti-target' modalities of BsAb for therapeutic efficacy. Several BsAb induce effective cytotoxicity as well as 'vaccine effects' in vivo. Here, Annemiek van Spriel and colleagues discuss how these results have catalysed renewed efforts to translate BsAb concepts into effective therapies.

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.

A single injection of polyethylene-glycol granulocyte colony-stimulating factor strongly prolongs survival of mice with systemic candidiasis.

Systemic candidiasis is a life-threatening disease occurring in immunocompromized patients. Granulocyte colony-stimulating factor (G-CSF) reduces mortality in experimental invasive candidiasis. Covalent conjugation of polyethylene-glycol (peg) to proteins increases their stability and in vivo bioactivity. In this study, the effect of a single subcutaneous injection of peg-G-CSF on lethal candidiasis was assessed. This was performed in acute and chronic candidiasis models in non-neutropenic FVB/N mice. Peg-G-CSF rapidly increased circulating polymorphonuclear leukocyte (PMNL) numbers in mice, sustaining high for >4 days. Candida albicans outgrowth from kidneys of infected mice was strongly reduced after peg-G-CSF treatment (5.76 log cfu/g kidney vs 7.66 control), with absence of hyphal outgrowth and enhanced PMNL influx. Moreover, peg-G-CSF increased survival of C. albicans -infected mice, whereas efficacy of uncoupled G-CSF was obtained only after repeated treatment. These data document a potent in vivo biological effect of peg-G-CSF, resulting in strongly enhanced resistance against systemic candidiasis.

Human immunoglobulin A receptor (FcalphaRI, CD89) function in transgenic mice requires both FcR gamma chain and CR3 (CD11b/CD18).

Even though more immunoglobulin A (IgA) is produced in humans than all other isotypes combined, relatively little is known about receptors that bind the Fc part of IgA. The myeloid IgA receptor, FcalphaRI (CD89), triggers various effector functions in vitro, but its in vivo role remains unclear. Here, a transgenic mouse model is described in which FcalphaRI is expressed under its own regulatory sequences. Receptor expression and regulation by cytokines was comparable to the human situation and hFcalphaRI can trigger phagocytosis and lysis of tumor cells. To analyze the contribution of the FcR gamma chain or the beta2 integrin CR3 (CD11b/CD18) in FcalphaRI biological function, FcalphaRI transgenic mice were crossed with either FcR gamma chain -/- or CR3 -/- mice. In contrast to in vitro data, FcR gamma chain was essential for surface expression of hFcalphaRI in vivo. Functional studies in hFcalphaRI/ gamma-/-mice were, therefore, limited. In vitro studies showed FcR gamma chain to be necessary for phagocytosis. Neither hFcalphaRI expression nor phagocytosis, triggered via hFcalphaRI, were influenced by CR3. Remarkably, the capacity to lyse tumor targets was ablated in hFcalphaRI transgenic/ CR3-/- mice, although binding of neutrophils to tumor cells was intact. This shows a previously unrecognized importance of CR3 for hFcalphaRI-mediated antibody-dependent cellular cytotoxicity (ADCC).

Effective phagocytosis and killing of Candida albicans via targeting FcgammaRI (CD64) or FcalphaRI (CD89) on neutrophils.

Invasive fungal infections are an increasing problem for immunocompromised patients. As an approach to improve targeting of polymorphonuclear leukocytes (PMNL) toward Candida albicans, the effect of bispecific antibodies (BsAbs) directed against C. albicans and either FcalphaRI or FcgammaRI was evaluated. Control PMNL and in vivo granulocyte colony-stimulating factor (G-CSF)-primed PMNL served as effector cells. A new radiometric killing assay for measuring candidacidal activity was developed to facilitate quantification of PMNL-mediated killing of C. albicans. BsAbs directed to either FcgammaRI (CD64) or FcalphaRI (CD89) on human PMNL effectively enhanced both phagocytosis and killing of C. albicans in vitro. Fungicidal activity triggered via FcgammaRI required in vivo priming with G-CSF, whereas FcalphaRI-mediated activity was not dependent on this growth factor. Furthermore, PMNL from human FcgammaRI-transgenic mice effectively phagocytosed and eliminated C. albicans in the presence of BsAbs. These results document the capacity of FcR-directed BsAbs and G-CSF to trigger antifungal immune responses.

The truncated estrogen receptor alpha variant lacking exon 5 is not involved in progesterone receptor expression in meningiomas.

Human meningioma tissues are mostly estrogen receptor (ER) negative and progesterone receptor (PR) positive in ligand binding and enzyme immuno assays. To explain this apparently ER independent PR expression, we investigated the existence of a 'hidden' ER variant, which would be capable of activating transcription of the PR gene. Total RNA of seven meningiomas, two breast cancer tissues and of MCF7 cells was analyzed by RT-PCR using primers situated in exon 4 and exon 6. Differential hybridization of the PCR transcripts with probes in exon 4 and 5 respectively, revealed a wild type ER (wtER) fragment and an exon 5 deleted ER variant (ERDelta5). PCR products of two meningiomas were cloned for sequence analysis. The result confirmed the existence of a wtER and ERDelta5.RT-PCR followed by Southern analysis was performed on mRNA of 23 meningiomas to determine the amount of ERDelta5 relative to wtER, which was compared to the PR content of the tissues. In contrast to our initial hypothesis and literature data on breast cancer, there was no relationship between the ERDelta5/wtER ratio and PR protein concentration. It is therefore concluded that ERDelta5 mRNA does not play the dominant role in PR synthesis in meningioma tissue.

Transforming growth factor-beta levels in maternal milk and expression in postnatal rat duodenum and ileum.

After birth, the gastrointestinal tract of the neonate is exposed to food and bacterial and environmental antigens. Maternal milk components may play a role in regulation of mucosal immune activity to luminal antigens. In this study we determine the ontogeny of transforming growth factor (TGF)-beta1-producing cells in the rat pup small intestine and assess maternal milk concentrations of TGF-beta. Intestinal tissue samples of duodenum and ileum were collected, processed, and stained for TGF-beta1, and in situ hybridization for TGF-beta1 mRNA was also performed on the duodenum. TGF-beta levels in milk were assayed by ELISA. TGF-beta2 levels in milk were high at d 6, and declined thereafter at d 10 and 19. TGF-beta1 was not detected. In contrast, the cell number and intensity of staining of TGF-beta1 peptide in the small intestine was low in 3- and 10-d-old rats and increased markedly by 19 d of life. In the duodenum mRNA levels mirrored this trend. TGF-beta1 expression in the lamina propria was absent before d 19, and increased progressively over time. Maternal milk TGF-beta2 levels are high in early milk and decrease during the weaning period. In contrast, endogenous TGF-beta production in the small intestine increases during the weaning period.

FcalphaRI (CD89) as a novel trigger molecule for bispecific antibody therapy.

Promising results from clinical trials with unconjugated antibodies stimulated renewed interest in immune effector mechanisms of monoclonal antibodies (MoAbs). We investigated the potential of IgA as antibody isotype for cell- or complement-mediated tumor cell lysis and assessed the potential of its myeloid Fc receptor, FcalphaRI (CD89), as trigger molecule for bispecific antibody (BsAb)-mediated immunotherapy. Comparing hapten-directed antibodies of human IgA2 with IgG1 or IgG3 isotypes, we found all three to mediate effective killing of sensitized tumor target cells in whole blood assays. Analysis of effector mechanisms showed IgG-mediated lysis to be predominantly complement-dependent, whereas IgA-dependent killing was primarily effector cell-mediated. A comparison of effector cell populations in antibody-dependent cell-mediated cytotoxicity (ADCC) showed neutrophils to be most important for IgA-dependent tumor cell killing, involving FcalphaRI as shown with Fc receptor blocking antibodies. Reverse ADCC experiments against target cells sensitized with Fc receptor antibodies, or assays with FcalphaRI-directed bispecific antibodies confirmed FcalphaRI as effective trigger molecule in polymorphonuclear neutrophil (PMN)-mediated lysis. During granulocyte colony-stimulating factor (G-CSF ) therapy, (FcalphaRI x HER-2/neu) bispecific antibodies induced enhanced killing of HER-2/neu positive SK-BR-3 breast cancer cells in whole blood assays. This enhanced cytotoxicity was paralleled by increased PMN counts, which lead to higher effector to target cell ratios in G-CSF-primed blood. Furthermore, bispecific antibodies, directed to FcalphaRI and Candida albicans, enhanced neutrophils' phagocytosis of fungi. In summary, these results identify IgA as an effective antibody isotype for immunotherapy, working primarily via FcalphaRI on neutrophils. They suggest FcalphaRI-directed bispecific antibodies and G-CSF to be an attractive combination for malignant or infectious diseases.