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Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca++ influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels. The CR1-elicited RBC Ca++ influx is accompanied by an increase in RBC membrane deformability that positively correlates with the number of preexisting CR1 molecules on RBC membranes. Biochemically, ligation of RBC CR1 causes a significant increase in phosphorylation levels of β-spectrin that is inhibited by preincubation of RBCs with DMAT, a specific casein kinase II inhibitor. We hypothesize that the CR1-dependent increase in membrane deformability could be relevant for facilitating the transfer of CR1-bound particles from the RBCs to the hepatic and splenic phagocytes.

Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca++influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels. The CR1-elicited RBC Ca++influx is accompanied by an increase in RBC membrane deformability that positively correlates with the number of preexisting CR1 molecules on RBC membranes. Biochemically, ligation of RBC CR1 causes a significant increase in phosphorylation levels of β-spectrin that is inhibited by preincubation of RBCs with DMAT, a specific casein kinase II inhibitor. We hypothesize that the CR1-dependent increase in membrane deformability could be relevant for facilitating the transfer of CR1-bound particles from the RBCs to the hepatic and splenic phagocytes.

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.

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.

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.

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell–derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage–specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.

Although the SDF-1 (CXCL12)/CXCR4 axis is important for B-cell development, it is not yet clear to what extent CC chemokines might influence B lymphopoiesis. In the current study, we characterized CC chemokine receptor 5 (CCR5) expression and function of primary progenitor B-cell populations in human bone marrow. CCR5 was expressed on all bone marrow B cells at levels between 150 and 200 molecules per cell. Stimulation of bone marrow B cells with the CCR5-binding chemokine macrophage inflammatory protein 1β (MIP-1β; CCL4) did not cause chemotaxis, but CCL4 was able to trigger potent calcium mobilization responses and activation of the mitogen-activated protein kinase (MAPK) pathway in developing B cells. We also determined that CCR5-binding chemokines MIP-1α (CCL3), CCL4, and RANTES (CCL5), specifically by signaling through CCR5, could affect all progenitor B-cell populations through a novel mechanism involving heterologous desensitization of CXCR4. This cross-desensitization of CXCR4 was manifested by the inhibition of CXCL12-induced calcium mobilization, MAPK activation, and chemotaxis. These findings indicate that CCR5 can indeed mediate biologic responses of bone marrow B cells, even though these cell populations express low levels of CCR5 on their cell surface. Thus, by modulation of CXCR4 function, signaling through CCR5 may influence B lymphopoiesis by affecting the migration and maturation of B-cell progenitors in the bone marrow microenvironment.

Although the SDF-1 (CXCL12)/CXCR4 axis is important for B-cell development, it is not yet clear to what extent CC chemokines might influence B lymphopoiesis. In the current study, we characterized CC chemokine receptor 5 (CCR5) expression and function of primary progenitor B-cell populations in human bone marrow. CCR5 was expressed on all bone marrow B cells at levels between 150 and 200 molecules per cell. Stimulation of bone marrow B cells with the CCR5-binding chemokine macrophage inflammatory protein 1β (MIP-1β; CCL4) did not cause chemotaxis, but CCL4 was able to trigger potent calcium mobilization responses and activation of the mitogen-activated protein kinase (MAPK) pathway in developing B cells. We also determined that CCR5-binding chemokines MIP-1α (CCL3), CCL4, and RANTES (CCL5), specifically by signaling through CCR5, could affect all progenitor B-cell populations through a novel mechanism involving heterologous desensitization of CXCR4. This cross-desensitization of CXCR4 was manifested by the inhibition of CXCL12-induced calcium mobilization, MAPK activation, and chemotaxis. These findings indicate that CCR5 can indeed mediate biologic responses of bone marrow B cells, even though these cell populations express low levels of CCR5 on their cell surface. Thus, by modulation of CXCR4 function, signaling through CCR5 may influence B lymphopoiesis by affecting the migration and maturation of B-cell progenitors in the bone marrow microenvironment.