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Complement-containing immune complexes can be presented to phagocytes by human erythrocytes bearing complement receptor 1 (CR1). Although this has long been assumed to be a mechanism by which humans are able to protect themselves from "extracellular" bacteria such as pneumococci, there is little direct evidence. In these studies we have investigated this question by comparing results for erythrocytes from transgenic mice expressing human CR1 on their erythrocytes to the results for wild-type mouse erythrocytes that do not express CR1. We demonstrate that human CR1 expression on murine erythrocytes allows immune adherence to beads opsonized with either mouse or human serum as a source of complement. The role of CR1 in immune adherence was supported by studies showing that it was blocked by the addition of antibody to human CR1. Furthermore, human CR1 expression enhances the immune adherence of opsonized pneumococci to erythrocytes in vitro, and the pneumococci attached to erythrocytes via CR1 can be transferred in vitro to live macrophages. Even more importantly, we observed that if complement-opsonized pneumococci are injected intravenously with CR1(+) mouse erythrocytes into wild-type mice (after a short in vitro incubation), they are cleared faster than opsonized pneumococci similarly injected with wild-type mouse erythrocytes. Finally, we have shown that the intravenous (i.v.) injection of pneumococci into CR1(+) mice also results in more rapid blood clearance than in wild-type mice. These data support that immune adherence via CR1 on erythrocytes likely plays an important role in the clearance of opsonized bacteria from human blood.

The ulcer-causing gastric pathogen Helicobacter pylori is the only bacterium known to colonize the harsh acidic environment of the human stomach. H. pylori survives in acidic conditions by producing urease, which catalyzes hydrolysis of urea to yield ammonia thus elevating the pH of its environment. However, the manner in which H. pylori is able to swim through the viscoelastic mucus gel that coats the stomach wall remains poorly understood. Previous rheology studies on gastric mucin, the key viscoelastic component of gastric mucus, indicate that the rheology of this material is pH dependent, transitioning from a viscous solution at neutral pH to a gel in acidic conditions. Bulk rheology measurements on porcine gastric mucin (PGM) show that pH elevation by H. pylori induces a dramatic decrease in viscoelastic moduli. Microscopy studies of the motility of H. pylori in gastric mucin at acidic and neutral pH in the absence of urea show that the bacteria swim freely at high pH, and are strongly constrained at low pH. By using two-photon fluorescence microscopy to image the bacterial motility in an initially low pH mucin gel with urea present we show that the gain of translational motility by bacteria is directly correlated with a rise in pH indicated by 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH sensitive fluorescent dye. This study indicates that the helicoidal-shaped H. pylori does not bore its way through the mucus gel like a screw through a cork as has previously been suggested, but instead achieves motility by altering the rheological properties of its environment.

Decay-accelerating factor (DAF, also known as CD55), a glycosylphosphatidylinositol-linked (GPI-linked) plasma membrane protein, protects autologous cells from complement-mediated damage by inhibiting complement component 3 (C3) activation. An important physical property of GPI-anchored complement regulatory proteins such as DAF is their ability to translate laterally in the plasma membrane. Here, we used single-particle tracking and tether-pulling experiments to measure DAF lateral diffusion, lateral confinement, and membrane skeletal associations in human erythrocyte membranes. In native membranes, most DAF molecules exhibited Brownian lateral diffusion. Fluid-phase complement activation caused deposition of C3b, one of the products of C3 cleavage, onto erythrocyte glycophorin A (GPA). We then determined that DAF, C3b, GPA, and band 3 molecules were laterally immobilized in the membranes of complement-treated cells, and GPA was physically associated with the membrane skeleton. Mass spectrometry analysis further showed that band 3, alpha-spectrin, beta-spectrin, and ankyrin were present in a complex with C3b and GPA in complement-treated cells. C3b deposition was also associated with a substantial increase in erythrocyte membrane stiffness and/or viscosity. We therefore suggest that complement activation stimulates the formation of a membrane skeleton-linked DAF-C3b-GPA-band 3 complex on the erythrocyte surface. This complex may promote the removal of senescent erythrocytes from the circulation.

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-gamma, and G protein-coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

The primary identified function of complement receptor 1 (CR1/CD35) on primate erythrocytes is to bind complement-tagged inflammatory particles including microbes and immune complexes. When erythrocytes circulate through liver and spleen, sinusoidal phagocytes remove CR1-adherent particles and erythrocytes return to the circulation. This process of immune adherence clearance is important for host defense and prevention of autoimmunity. CR1 was previously described as clustered in the human erythrocyte membrane, which was thought to be necessary for binding complement-opsonized particles. In contrast, we demonstrate that on erythrocytes CR1 is not clustered, but dispersed, and able to bind complement-tagged particles. When fresh erythrocytes are solubilized by nonionic detergent, CR1 partitions to the cytoskeleton fraction. Using a PDZ-peptide array, CR1's cytoplasmic tail, which contains 2 PDZ-motifs, binds PDZ domains 2, 3, and 5 of Fas-associated phosphatase 1 (FAP-1), a scaffolding protein. We show that FAP-1, not previously recognized as an erythroid protein, is expressed on circulating erythrocytes. CR1 and FAP-1 coimmunoprecipitate, which confirms their molecular association. Disperse CR1 on erythrocytes may be advantageous for capturing immune-complexes, while ligation-induced CR1 clustering may prevent ingestion of the erythrocyte during the immune-complex transfer to the macrophages by keeping the opsonic stimulus localized thus preventing phagocyosis.

Human red blood cells (RBCs) exhibit vibratory motions, referred to as "flickering." Their dynamical properties, classically attributed to thermal mechanisms, have not been fully characterized. Using detrended fluctuation analysis and multiscale entropy methods, we show that the short-term flickering motions of RBCs, observed under phase contrast microscopy, have a fractal scaling exponent close to that of 1f noise and exhibit complex patterns over multiple time scales. Further, these dynamical properties degrade with in vivo aging such that older cells that have been in the circulation longer generate significantly (p<0.003) less complex flickering patterns than newly formed cells. Quantitative assessment of multiscale flickering may provide a way of measuring RBC functionality. Membrane models need to account for the complex properties of these motions and their changes with in vivo senescence.

The capacity of airway eosinophils, potentially pertinent to allergic diseases of the upper and lower airways, to function as professional APCs, those specifically able to elicit responses from unprimed, Ag-naive CD4(+) T cells has been uncertain. We investigated whether airway eosinophils are capable of initiating naive T cell responses in vivo. Eosinophils, isolated free of other APCs from the spleens of IL-5 transgenic mice, following culture with GM-CSF expressed MHC class II and the costimulatory proteins, CD40, CD80, and CD86. Eosinophils, incubated with OVA Ag in vitro, were instilled intratracheally into wild-type recipient mice that adoptively received i.v. infusions of OVA Ag-specific CD4(+) T cells from OVA TCR transgenic mice. OVA-exposed eosinophils elicited activation (CD69 expression), proliferation (BrdU incorporation), and IL-4, but not IFN-gamma, cytokine production by OVA-specific CD4(+) T cells in paratracheal lymph nodes (LN). Exposure of eosinophils to lysosomotropic NH(4)Cl, which inhibits Ag processing, blocked each of these eosinophil-mediated activation responses of CD4(+) T cells. By three-color fluorescence microscopy, OVA Ag-loaded eosinophil APCs were physically interacting with naive OVA-specific CD4(+) T cells in paratracheal LN after eosinophil airway instillation. Thus, recruited luminal airway eosinophils are distinct allergic "inflammatory" professional APCs able to activate primary CD4(+) T cell responses in regional LNs.

Measurement of erythrocyte [red blood cells (RBC)] complement receptor type 1 (CR1, CD35) has the potential to serve as a sensitive assessment of complement activation and immune complex clearance. All previously reported monoclonal antibodies (MoAb) to the extracellular region of CR1 recognize epitopes within the long homologous repeats (LHR) of CR1 and the epitopes for the most frequently used MoAbs are repeated at least twice per CR1 molecule. Furthermore, CR1 exhibits structural polymorphism characterized by a variable number of LHR per molecule. Thus, accurate enumeration of cell surface CR1 using currently available MoAb would require that the results be corrected for the number of antibody epitopes per CR1 molecule encoded by each individual's alleles. To obtain a MoAb to a non-polymorphic epitope on human CR1, hybridomas were generated from mice immunized with recombinant soluble CR1 (sCR1) and MoAb were screened for those that recognized the full-length extracellular domain but failed to bind to all four recombinant LHR fragments. A single antibody, CR1-2B11, was identified and was found to recognize an epitope located wholly within SCR29-30 of CR1, NH2-terminal to an elastase cleavage site. Like other CR1 MoAb, the CR1-2B11 epitope expression decreased on old erythrocytes compared to younger cells and CR1-2B11 did not identify a CR1 'stump' on RBC. Importantly, CR1-2B11 immunofluorescence did not change with storage or handling of RBC, unlike the apparent decrease in immunofluorescence observed with other MoAb. CR1-2B11 should be useful for the accurate enumeration of RBC CR1.

Stromal cells isolated from bone marrow (BMSCs), often referred to as mesenchymal stem cells, are currently under investigation for a variety of therapeutic applications. However, limited data are available regarding receptors that can influence their homing to and positioning within the bone marrow. In the present study, we found that second passage BMSCs express a unique set of chemokine receptors: three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). BMSCs cultured in serum-free medium secrete several chemokine ligands (CCL2, CCL4, CCL5, CCL20, CXCL12, CXCL8, and CX3CL1). The surface-expressed chemokine receptors were functional by several criteria. Stimulation of BMSCs with chemokine ligands triggers phosphorylation of the mitogen-activated protein kinase (e.g., extracellular signal-related kinase [ERK]-1 and ERK-2) and focal adhesion kinase signaling pathways. In addition, CXCL12 selectively activates signal transducer and activator of transcription (STAT)-5 whereas CCL5 activates STAT-1. In cell biologic assays, all of the chemokines tested stimulate chemotaxis of BMSCs, and CXCL12 induces cytoskeleton F-actin polymerization. Studies of culture-expanded BMSCs, for example, 12-16 passages, indicate loss of surface expression of all chemokine receptors and lack of chemotactic response to chemokines. The loss in chemokine receptor expression is accompanied by a decrease in expression of adhesion molecules (ICAM-1, ICAM-2, and vascular cell adhesion molecule 1) and CD157, while expression of CD90 and CD105 is maintained. The change in BMSC phenotype is associated with slowing of cell growth and increased spontaneous apoptosis. These findings suggest that several chemokine axes may operate in BMSC biology and may be important parameters in the validation of cultured BMSCs intended for cell therapy.

Secretion of interleukin-4 (IL-4) by leukocytes is important for varied immune responses including allergic inflammation. Within eosinophils, unlike lymphocytes, IL-4 is stored in granules (termed specific granules) and can be rapidly released by brefeldin A (BFA)-inhibitable mechanisms upon stimulation with eotaxin, a chemokine that activates eosinophils. In studying eotaxin-elicited IL-4 secretion, we identified at the ultrastructural level distinct vesicular IL-4 transport mechanisms. Interleukin-4 traffics from granules via two vesicular compartments, large vesiculotubular carriers, which we term eosinophil sombrero vesicles (EoSV), and small classical spherical vesicles. These two vesicles may represent alternative pathways for transport to the plasma membrane. Loci of both secreted IL-4 and IL-4-loaded vesicles were imaged at the plasma membranes by a novel EliCell assay using a fluoronanogold probe. Three dimensional electron tomographic reconstructions revealed EoSVs to be folded, flattened and elongated tubules with substantial membrane surfaces. As documented with quantitative electron microscopy, eotaxin-induced significant formation of EoSVs while BFA pretreatment suppressed eotaxin-elicited EoSVs. Electron tomography showed that both EoSVs and small vesicles interact with and arise from granules in response to stimulation. Thus, this intracellular vesicular system mediates the rapid mobilization and secretion of preformed IL-4 by activated eosinophils. These findings, highlighting the participation of large tubular carriers, provide new insights into vesicular trafficking of cytokines.

Complement-opsonized particles become immune adherent to complement receptor 1 (CR1 or CD35) on human erythrocytes, allowing particles to be ingested by phagocytes in the liver and the spleen. We investigated the role that immune adherence plays in the uptake and killing of Salmonella montevideo by human neutrophils. Exposure to serum induced loss of flagella and facilitated immune adherence, which was followed by more-efficient phagocytosis and killing, compared with that after exposure to serum-opsonized, free bacteria. One correlate of bacterial killing is the fusion of phagosomes with lysosomes, which can be monitored by Lyso-Tracker or lysosomal-associated membrane protein 2 colocalization with the intracellular bacteria. At 5 min, phagolysosmal fusion was significantly faster for immune-adherent bacteria than for non-immune-adherent bacteria, but, by 35 min, the difference between the 2 groups was minimal. Immune adherence also facilitated the ingestion of antibody complement-opsonized fluorescent microspheres, but, unlike bacteria, most internalized microspheres failed to fuse with lysosomes. However, addition of lipopolysaccharide, a Toll-like receptor ligand, to microspheres directed their intracellular trafficking, resulting in rapid lysosomal fusion. Thus, immune adherence facilitates phagocytosis, but the route of intracellular processing depends on the molecular nature of the target and is independent of host complement and antibody.

Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage PhiX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics.

Calreticulin, a candidate C1q receptor, was shown recently to be present on the surface of human neutrophils in association with glycosylphosphatidylinositol (GPI) anchored proteins, particularly CD59. In this study, we show that antibodies to CD59, as well as to every other GPI-anchored protein tested, inhibited the C1q-triggered release of O(2)(-) from PMN. Methyl beta cyclodextrin (M beta CD) treatment of the cells to disrupt lipid rafts also prevented C1q-triggered O(2)(-) production. beta(2) integrin-dependent co-stimulation is required for O(2)(-) production from PMN, however M beta CD had no effect on LFA-1 or Mac-1-mediated adhesion, soluble iC3b binding to PMN, or spreading and migration, all of which suggested that PMN integrin function remained intact. Flow cytometric analysis of PMN treated with M beta CD showed upregulation of PMN granule-associated integrins and a corresponding increase in integrin activation-reporter epitopes, in contrast to the decreased expression of GPI-anchored antigens. These data support a model where lipid rafts and their associated GPI-anchored proteins are critical for C1q-triggered O(2)(-) production, consistent with a model where calreticulin serves as the C1q receptor for O(2)(-) production from PMN.

Calreticulin, which has been proposed to be a C1q receptor on neutrophils, has neither a transmembrane domain nor a GPI-anchor attachment site and must utilize an adaptor molecule to attach to the plasma membrane. The expression of ecto-calreticulin on purified human neutrophils did not result from contamination by soluble or intracellular calreticulin released during cell fractionation because it was expressed on circulating neutrophils, and the expression did not increase significantly with neutrophil isolation. All neutrophils expressed calreticulin with a unimodal distribution. Treatment of neutrophils with either a cholesterol-binding agent or phosphatidylinositol-specific phospholipase C dramatically decreased ecto-calreticulin expression indicating that the adaptor molecule(s) are located in lipid rafts and have a GPI-anchor. Analysis for the co-expression of specific GPI-anchored proteins and ecto-calreticulin in cells that were deficient in specific GPI-anchored proteins, indicated that ecto-calreticulin was best associated with CD59. Calreticulin reciprocally immunoprecipited with CD59, which provided direct evidence that CD59 is an adaptor for ecto-calreticulin. Immunofluorescence and confocal microscopy demonstrated that ecto-calreticulin co-localized with a fraction of CD59 at the cell surface. Cross-linking ecto-calreticulin with antibodies induced a Ca2+ flux, which suggests that ecto-calreticulin is capable of signaling following ligand binding. Ecto-calreticulin has been associated with diverse cellular functions. An appreciation that the adaptors for ecto-calreticulin are GPI-anchored will provide a framework for understanding any common features underlying ecto-calreticulin ligation.

Eosinophils contain several preformed cytokines within their specific granules. Therefore, without requiring them in de novo synthesis of cytokines, eosinophils can release quantities of granule-derived cytokines by highly regulated mechanisms. However, eosinophil "degranulation" is poorly understood, in part, because available methodologies did not appear appropriate for analyzing vesicular mobilization and transport of eosinophil granular contents. The EliCell assay is a microscopic methodology substantially modified from other techniques employed to detect cytokine release (i.e., ELISPOT). The method is a dual antibody capture/detection system in which viable eosinophils are incubated in a solid streptavidin-conjugated agarose matrix, which contains a biotinylated capture antibody against the cytokine of interest. Released cytokine is detected around non-permeabilized eosinophils with a separate fluorochrome-labeled detection antibody. Thus, the EliCell system captures and detects extracellular cytokines at the site of their release from eosinophils. As examples, we have used EliCell essays to detect the selective release of either IL-4 or IL-12 cytokines found preformed in eosinophils-from eotaxin- or anti-CD9-stimulated eosinophils, respectively. With appropriate pairs of antibodies, any preformed cytokine found into eosinophil granules could be studied and the mechanisms of their secretion evaluated by using the EliCell assay.

C1q is the recognition unit of the first component of complement that binds not only IgG and IgM containing immune complexes, but also recognizes foreign structures such as the lipid A of endotoxin, and molecules expressed at the surface of apoptotic cells. In this review, the plasma membrane receptors and binding proteins for C1q are discussed and new data are presented on calreticulin expression on human peripheral blood cells. Although much is known about C1q receptors and binding molecules there are still many questions regarding their role in vivo.

Mannan-binding lectin (MBL), a member of the collectin family, is known to have opsonic function, although identification of its cellular receptor has been elusive. Complement C1q, which is homologous to MBL, binds to complement receptor 1 (CR1/CD35), and thus we investigated whether CR1 also functions as the MBL receptor. Radioiodinated MBL bound to recombinant soluble CR1 (sCR1) that had been immobilized on plastic with an apparent equilibrium dissociation constant of 5 nM. N-acetyl-d-glucosamine did not inhibit sCR1-MBL binding, indicating that the carbohydrate binding site of MBL is not involved in binding CR1. C1q inhibited MBL binding to immobilized sCR1, suggesting that MBL and C1q might bind to the same or adjacent sites on CR1. MBL binding to polymorphonuclear leukocytes (PMNs) was associated positively with changes in CR1 expression induced by phorbol myristate acetate. Finally, CR1 mediated the adhesion of human erythrocytes to immobilized MBL and functioned as a phagocytic receptor on PMNs for MBL-immunoglobulin G opsonized bacteria. Thus, MBL binds to both recombinant sCR1 and cellular CR1, which supports the role of CR1 as a cellular receptor for the collectin MBL.

Eosinophils contain many preformed cytokines and chemokines, which are stored in specific granules along with cationic granule proteins. Mobilization and release of these granule contents can be selective and mediated by vesicular transport. We have developed a sensitive method to detect and quantitate eosinophil vesicular transport-mediated release of specific eosinophil proteins. Our EliCell assay is based on microscopic observations of individual viable eosinophils embedded in an agarose matrix that contains immobilized antibody to the protein of interest. Following stimulation of eosinophils, released protein is bound by the capture antibody at its site of release and is detected by a fluorochrome-conjugated detection antibody. We have validated this assay by evaluating interferon-gamma-induced release of RANTES from eosinophils. Extracellularly released RANTES was visualized as focal immunoflourescent staining and was quantitated by scoring the numbers of eosinophils releasing RANTES and by measuring the fluorescent intensity over individual eosinophils. In comparison with ELISA assays of RANTES released into supernatant fluids by interferon-gamma-stimulated eosinophils, EliCell assays were more sensitive enabling detection of RANTES release at earlier times and at lower levels of interferon-gamma stimulation. The EliCell assay provides a sensitive method to study the regulated release of eosinophil-derived cytokines, chemokines and other granule proteins.