Release of iC3b from apoptotic tumor cells induces tolerance by binding to immature dendritic cells in vitro and in vivo

Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Apoptotic cells are known to activate homologous complement and to be opsonized with iC3b. Since maturation of dendritic cells (DC) can be inhibited by binding of iC3b to the complement receptor 3 (CR3, CD11b/CD18) and because immature DC induce tolerance, we investigated the induction of tolerance after pulsing DC with apoptotic cells in the presence or absence of native serum. Apoptosis in pancreatic carcinoma cells was induced either by heat-stress, chemotherapy or anti-Her2 antibody. Monocyte-derived DC were pulsed with apoptotic cells with or without native serum. DC were analyzed for the maturation state by flow cytometry and the cytotoxic activity was determined. Tolerance was prevented by addition of substances such as anti CD11b or N–acetyl-D-Glucosamine (NADG) which block iC3b binding to CR3. Furthermore, binding of iC3b from apoptotic cells to DC was blocked in a syngeneic pancreatic carcinoma mouse model. All of the former strategies for apoptosis induction resulted in iC3b release. Pulsing DC with apoptotic cells in the presence of serum prevents maturation of DC and induces finally tolerance. This tolerance could be prevented almost completely by blocking the interaction of iC3b with the CR3 receptor. This could be shown as well in an immunocompetent mouse model. Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Release of iC3b from apoptotic tumor cells prevents fully maturation of DC and immature DC induce antigen-specific silencing or tolerance. Blocking of iC3b-binding could mostly prevent this effect.     

The class A macrophage scavenger receptor type I (SR-AI) recognizes complement iC3b and mediates NF-κB activation

The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis. In addition, SR-AI modulates macrophage activation through cell signaling. However, investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors. Therefore, we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling. On 293T cells, SR-AI could respond to E. coli DH5α, leading to NF-κB activation and IL-8 production. However, this requires E. coli DH5α to be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion. Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5α and blocks DH5α stimulation of SR-AI signaling. Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b. By mutagenesis, The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α. These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.     

Gram-negative bacteria and phagocytic cell interaction mediated by complement receptor 3

Complement receptor 3 (CR3) is an integrin that recognizes several different ligands. Binding to CR3 in phagocytic cells activates signaling pathways involved in cytoskeleton rearrangement, regulation of cell motility, alteration of gene expression and phagocytosis of complement-opsonized as well as of some non-opsonized particles and pathogenic bacteria. However, CR3-mediated phagocytosis of some Gram-negative bacteria does not induce bacterial clearance. Pseudomonas aeruginosa, Salmonella and Escherichia coli are eliminated after phagocytic cell-bacteria interaction mediated by CR3. However, Bordetella takes advantage of the CR3 function and uses it to enter into macrophages leading to bacterial survival. The final fate of the pathogen is determined by combinations of host and bacterial factors, in which molecular interactions between CR3 and bacterial ligands are involved.     

Complement receptor 3 (CD11b/CD18) mediates type I and type II phagocytosis during nonopsonic and opsonic phagocytosis,respectively

Two types of opsonic phagocytosis have been defined depending on the receptor engaged: FcgammaRs mediate type I phagocytosis of IgG-coated particles; complement receptor 3 (CR3) mediates type II phagocytosis of complement-coated particles. In addition to opsonic phagocytosis, CR3 also mediates nonopsonic phagocytosis of zymosan (Z) and Mycobacterium kansasii through engagement of distinct sites. Using Chinese hamster ovary cells stably expressing human CR3, we studied CR3-mediated ingestion of nonopsonized particles, Z or M. kansasii, compared with opsonized zymosan (OZ). We show that 1) while OZ sinks into cells, Z is engulfed by pseudopodia as visualized by electron microscopy; 2) in contrast to OZ, nonopsonic phagocytosis of Z and M. kansasii depends on Rac and Cdc42 but not on Rho activity; and 3) CR3-mediated phagocytosis of Z depends on the kinase activity of the Src family tyrosine kinase Hck, while OZ internalization does not. Therefore, CR3 mediates type I phagocytosis under nonopsonic conditions and type II under opsonic conditions. This is the first evidence that a single receptor can mediate both types of phagocytosis depending on the ligand used.     

An urokinase receptor antagonist that inhibits cell migration by blocking the formyl peptide receptor

Urokinase receptor (uPAR) plays a key role in physiological and pathological processes sustained by an altered cell migration. We have developed peptides carrying amino acid substitutions along the Ser(88)-Arg-Ser-Arg-Tyr(92) (SRSRY) uPAR chemotactic sequence. The peptide pyro glutamic acid (pGlu)-Arg-Glu-Arg-Tyr-NH2 (pERERY-NH(2)) shares the same binding site with SRSRY and competes with N-formyl-Met-Leu-Phe (fMLF) for binding to the G-protein-coupled N-formyl-peptide receptor (FPR). pERERY-NH(2) is a dose-dependent inhibitor of both SRSRY- and fMLF-directed cell migration, and prevents agonist-induced FPR internalization and fMLF-dependent ERK1/2 phosphorylation. pERERY-NH(2) is a new and potent uPAR inhibitor which may suggest the generation of new pharmacological treatments for pathological conditions involving increased cell migration.     

CR1, the C3b receptor, mediates binding of infective Leishmania major metacyclic promastigotes to human macrophages

The role of complement receptors on monocyte derived human macrophages in phagocytosis of infective (MP) and noninfective (LP) developmental stages of Leishmania major promastigotes was studied. We compared binding of these specific developmental stages to MO after preincubation in fresh or heat-inactivated serum. Although LP do not require fresh serum for attachment, MP were dependent on serum C opsonization for entry. Inhibition of CR1 substantially abolished binding of the infective MP. In contrast, inhibition of iC3bR (CR3 and p150,95) had no effect on MP binding. Inhibition of both iC3bR, however, did block binding of nonopsonized LP. Attachment of LP to CR3 was blocked by fluid phase addition of mAb OKM1 and M1/70, which inhibit complement-independent binding to CR3, but not by mAb OKM10 which inhibits iC3b binding to this receptor. After fresh serum pretreatment of LP, however, only simultaneous inhibition of CR3 and CR1 effectively blocked their attachment. Addition of mannan did not inhibit attachment of either promastigote stage. Both opsonized and nonopsonized LP trigger a respiratory burst in MO, possibly via the C independent site in CR3, whereas the CR1-mediated uptake of MP does not generate a respiratory burst. The use of this receptor by MP may facilitate their subsequent intracellular survival.     

A role for mammalian diaphanous-related formins in complement receptor (CR3)-mediated phagocytosis in macrophages

Macrophages, dendritic cells, and neutrophils use phagocytosis to capture and clear off invading pathogens. The process is triggered by the interaction of ligands on the pathogens' surface with specific phagocytic receptors, including immunoglobulin (FcR) and complement C3bi (CR3) receptors (integrin alpha(M)beta2, Mac1) . Localized actin-filament assembly that acts as the driving force for particle engulfment is controlled by Rho-family small GTPases . RhoA regulates CR3-mediated phagocytosis through a mechanism that is still unclear . Mammalian Diaphanous-related (mDia) formins participate in the generation of a diverse set of actin-remodeling events downstream of RhoA , and mDia1 is recruited around fibronectin-coated beads in a RhoA-dependent manner in fibroblasts . Here, we set out to examine whether mDia proteins are involved in CR3-mediated phagocytosis in macrophages. We show that the RhoA effector mDia1 is recruited early during CR3-mediated phagocytosis and colocalizes with polymerized actin in the phagocytic cup. Interfering with mDia activity inhibits CR3-mediated phagocytosis while having no effect on FcR-mediated phagocytosis. These results indicate a new function for mDia proteins in the regulation of actin polymerization during CR3-mediated phagocytosis.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.     

Phagocytosis Is the Main CR3-Mediated Function Affected by the Lupus-Associated Variant of CD11b in Human Myeloid Cells

The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis.     

Sodium fluoride reveals multiple pathways for regulation of surface expression of the C3b/C4b receptor (CR1) on human polymorphonuclear leukocytes

We have examined the effects of NaF on C3b receptor (CR1) expression and function in human polymorphonuclear leukocytes (PMN). Plasma membrane expression of CR1 was determined with a monoclonal antibody (3D9); CR1 function was assessed with erythrocytes bearing C3b (EC3b) or C3b oligomers prepared with avidin and biotin. NaF inhibited in a dose-dependent manner CR1-mediated phagocytosis and NaF inhibited f-met-leu-phe or phorbol dibutyrate-induced increases in CR1 expression, with 50% inhibition at 5 mM NaF. Increased plasma membrane expression of CR3 induced by f-met-leu-phe also was inhibited by NaF. However, increased CR1 and CR3 expression due to incubation at 37 degrees C were unaffected by 10 mM NaF. Incubation of PMN with 10 mM NaF depleted 80% of intracellular adenosine triphosphate (ATP) after 10 min. However, inhibition of CR1 function was unrelated to ATP level, inasmuch as normal increases in CR1 expression and in phagocytosis occurred 20 min after removal of NaF, whereas ATP levels remained below 25% of normal. Strikingly, internalization of soluble oligomeric C3b ligands was unaffected by 10 mM NaF, which completely inhibited phorbol dibutyrate-induced CR1 internalization and EC3b phagocytosis. We conclude that there are two different mechanisms for increasing plasma membrane expression of CR1, one of which is inhibitable by NaF. Moreover, there are two distinct pathways of CR1 internalization which can also be distinguished by their sensitivity to NaF.     

Tumor-promoting phorbol esters induce rapid internalization of the C3b receptor via a cytoskeleton-dependent mechanism

Plasma membrane expression as well as phagocytic capability of the C3b receptor (CR1) are under regulatory control. Phorbol esters are one class of agents which have been shown to influence both of these events. In this study, by using radiolabeled Fab fragments of a monoclonal anti-CR1 antibody to tag the receptor and acid elution of surface-bound Fab, we showed that both phorbol myristate acetate and phorbol dibutyrate induced internalization of the C3b receptor; this occurred in a dose- and time-dependent manner in the absence of occupancy of the receptor by ligand. This was shown to occur in neutrophils, monocytes, and macrophages. We also showed that phorbol esters enhanced CR1-dependent phagocytosis despite the presence of two-thirds fewer receptors present on the plasma membrane. However, fibronectin, another agent that influences phagocytosis, had no effect on receptor internalization. Phorbol ester internalization was temperature-dependent and was inhibitable by cytochalasins B and D. Inhibition of internalization was reversible when cytochalasin B was removed. Phorbol esters also induced increased detergent insolubility of CR1 with kinetics similar to those of receptor internalization. It is possible that association of CR1 with the cytoskeleton is important to the process of "activation" of CR1 in phagocytosis.     

Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18).

C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.     

Interaction of two phagocytic host defense systems: Fcγ receptors and complement receptor 3

Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.     

Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.     

An essential role for talin during alpha(M)beta(2)-mediated phagocytosis

The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG- and C3bi-opsonized red blood cells in macrophages. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, alpha(M)beta(2) integrin) and not through the Fc gamma receptor. We show that talin physically interacts with CR3/alpha(M)beta(2) and that this interaction involves the talin head domain and residues W747 and F754 in the beta(2) integrin cytoplasmic domain. The CR3/alpha(M)beta(2)-talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/alpha(M)beta(2) binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/alpha(M)beta(2)-mediated phagocytosis, most noticeably activation of the CR3/alpha(M)beta(2) receptor and phagocytic uptake.     

Transcriptional Regulation of Urokinase Receptor in High-（95D） and Low-metastatic （95C） Human Lung Cancer Cells

The malignant neoplasm is characterized by progres-sive growth and the ability to disseminate tumor cellsbeyond the boundaries of the parent tumor. Tumor in-vasion and metastasis are important aspects of tumorprogression and the formation of tumor and metastasis is aprincipal contributing factor to cancer morbidity andmortality. During this process urokinase (urokinase-typeplasminogen activator, uPA) and its receptor (uPAR) playan important role. uPAR is a 55–60 kD glycoprotein which is a…     

A co-operative interaction between Neisseria gonorrhoeae and complement receptor 3 mediates infection of primary cervical epithelial cells

Little is known about the pathogenesis of gonococcal infection within the lower female genital tract. We recently described the distribution of complement receptor 3 (CR3) on epithelia of the female genital tract. Our studies further indicate that CR3-mediated endocytosis serves as a primary mechanism by which N. gonorrhoeae elicits membrane ruffling and cellular invasion of primary, human, cervical epithelial cells. We have extended these studies to describe the nature of the gonococcus-CR3 interaction. Western Blot analysis demonstrated production of alternative pathway complement components by ecto- and endocervical cells which allows C3b deposition on gonococci and its rapid conversion to iC3b. Anti-iC3b and -factor I antibodies significantly inhibited adherence and invasion of primary cervical cells, suggesting that iC3b covalently bound to the gonococcus serves as a primary ligand for CR3 adherence. However, gonococcal porin and pili also bound to the I-domain of CR3 in a non-opsonic manner. Binding of porin and pili to CR3 were required for adherence to and invasion of cervical epithelia. Collectively, these data suggest that gonococcal adherence to CR3 occurs in a co-operative manner, which requires gonococcal iC3b-opsonization, porin and pilus. In conjunction, these molecules facilitate targeting to and successful infection of the cervical epithelium.     

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex

Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.     

Cross-regulation of VPAC(2) receptor desensitization by M(3) receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

In gastrointestinal smooth muscle cells, VPAC(2) receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M(3) receptors regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC(2) receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M(2) receptor antagonist (i.e., activation of M(3) receptors) inhibited VPAC(2) receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M(3) receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M(2) receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC(2) receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G(s)-coupled receptor phosphorylation and internalization by G(q)-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.     

Cross-talk between CD14 and complement receptor 3 promotes phagocytosis of mycobacteria: regulation by phosphatidylinositol 3-kinase and cytohesin-1

The glycosylphosphatidyl anchored molecule CD14 to the monocyte membrane plays a prominent role in innate immunity, and the paradigms for CD14 selective signaling are beginning to be elucidated. In this study, transfected human monocytic cell line THP-1 and Chinese hamster ovary (CHO) fibroblastic cells were used to examine phagocytosis of Mycobacterium bovis bacillus Calmette-Guerin (BCG). Flow cytometry was combined with molecular and biochemical approaches to demonstrate a dual mechanism for BCG internalization involving either CD14 alone or a CD14-regulated complement receptor (CR)3-dependent pathway. Phagocytosis by CD14-positive THP-1 cells was attenuated by phosphatidylinositol-3 inhibitors LY294002 and wortmannin and experiments using transfected CHO cells showed substantial accumulation of phosphatidylinositol-3,4,5-trisphosphate at the BCG attachment site in CHO cells expressing CD14 and TLR2 suggesting that bacteria bind to CD14 and use TLR2 to initiate a PI3K signaling pathway. Additional experiments using blocking Abs showed that anti-TLR2 Abs inhibit phagocytosis of BCG by THP-1 cells. Furthermore, knockdown of cytohesin-1, a PI3K-regulated adaptor molecule for beta(2) integrin activation, specifically abrogated CD14-regulated CR3 ingestion of BCG consistent with the observation of physical association between CR3 and cytohesin-1 in cells stimulated with mycobacterial surface components. These findings reveal that mycobacteria promote their uptake through a process of "inside-out" signaling involving CD14, TLR2, PI3K, and cytohesin-1. This converts low avidity CR3 into an active receptor leading to increased bacterial internalization.