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Phagocytosis occurs primarily through two main processes in macrophages: the Fcγ receptor- and the integrin αMβ2-mediated processes. Complement C3bi-opsonized particles are known to be engulfed through integrin αMβ2-mediated process, which is regulated by RhoA GTPase. C3 toxin fused with Tat-peptide (Tat-C3 toxin), an inhibitor of the Rho GTPases, was shown to markedly inhibit the phagocytosis of serum (C3bi)-opsonized zymosans (SOZs). However, 8CPT-2Me-cAMP, an activator of exchange protein directly activated by cAMP (Epac, Rap1 guanine nucleotide exchange factor), restored the phagocytosis of the SOZs that was previously inhibited by the Tat-C3 toxin. In addition, a constitutively active form of Rap1 GTPase (CA-Rap1) also restored the phagocytosis that was previously reduced by a dominant negative form of RhoA GTPase (DN-RhoA). This suggests that Rap1 can replace the function of RhoA in the phagocytosis. Inversely, CA-RhoA rescued the phagocytosis that was suppressed by DN-Rap1. These findings suggest that both RhoA and Rap1 GTPases collectively regulate the phagocytosis of SOZs. In addition, filamentous actin was reduced by the Tat-C3 toxin, which was again restored by 8CPT-2Me-cAMP. Small interfering profilin suppressed the phagocytosis, suggesting that profilin is essential for the phagocytosis of SOZs. Furthermore, 8CPT-2Me-cAMP increased the co-immunoprecipitation of profilin with Rap1, whereas Tat-C3 toxin decreased that of profilin with RhoA. Co-immunoprecipitations of profilin with actin, Rap1, and RhoA GTPases were augmented in the presence of GTPγS rather than GDP. Therefore, we propose that both Rap1 and RhoA GTPases regulate the formation of filamentous actin through the interaction between actin and profilin, thereby collectively inducing the phagocytosis of SOZs in macrophages.

Several events accompany integrin-mediated phagocytosis by myeloid cells. These include local pseudopod and phagocytic cup formation followed by Ca(2+) signalling. However, there is also a role for localised phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P(3)] production. Here we report that in neutrophilic HL-60 cells expressing PH-Akt-GFP, binding of iC3b-coated zymosan particles (2 microm in diameter) via beta2 integrin induces an incomplete phagocytic cup to form before either PtdIns(3,4,5)P(3) or phosphatidylinositol (3,4) bisphosphate [PtdIns(3,4)P(2)] production or Ca(2+) signalling. These phosphoinositides then accumulated locally at the site of the phagocytic cup and Ca(2+) signalling and phagosome closure follows immediately. Although photobleaching showed that PH-Akt-GFP was freely diffusible in the cytosol and able to dissociate from the phagocytic cup, it was restricted to the plasma membrane of the formed but open phagosome and failed to diffuse into the surrounding plasma membrane or neighbouring phagocytic cups even if connected. Inhibition of phosphoinositide (PI) 3-kinase or depletion of membrane cholesterol inhibited both Ca(2+) signalling and phagosome closure, but had no effect on particle binding or phagocytic cup formation. We therefore conclude that PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2) generation was not required for the events that initiate the formation of the phagocytic cup, but that anchoring of PtdIns(3,4,5)P(3) at the phagocytic cup is an essential step for phagosome closure and Ca(2+) signalling.

Flow cytofluorimetry identifies and quantifies cell markers of different leukocyte subpopulations by combining cytofluorimetry with the differences in the light scattering properties of the leukocytes in mixed populations. In the phagocytic assay, reported in this paper, the experimental conditions were selected in such a way that it was possible to analyse the phagocytic function of granulocytes in peripheral blood without time-consuming cell separation. The percentage of phagocytosing granulocytes was not dependent on the concentration of granulocytes at the selected incubation time and particle (yeast-C3b) concentration. Furthermore, it was possible to adapt a previously described fluorescence quenching technique (FQ method) to differentiate between attachment and ingestion. Crystal violet, originally used in the FQ method, could not be used in this assay due to its lysomotropic effect. Trypan blue at a concentration of 0.25 mg/ml or higher at pH 4.5 showed a plateau effect in fluorescence quenching indicating an effect on attached but not ingested particles. This assay offers a simple technique to screen the functional properties of phagocytic cells in peripheral blood.

Monolayers of human peripheral blood monocytes in the absence of exogenous proteins ingest a variety of natural particulate activators of the human alternative complement pathway. Sheep erythrocytes, which do not ordinarily activate the human alternative complement pathway or initiate a direct monocyte phagocytic response, can be modified to exhibit both functions by the deletion or alteration of membrane sialic acid residues. Enzymatic removal of the sialic acid residues with sialidase or their conversion to heptulosonic acid derivatives by limited oxidation with NaIO4 and reduction with BH4- have equivalent dose-response effects on the capacity of the altered sheep erythrocytes to initiate the phagocytic response by human monocytes or to activate the alternative pathway in human serum. The deposition of C3b on native sheep erythrocytes had little effect on their ingestion by human monocytes, whereas the fixation of C3b on desialated sheep erythrocytes had a synergistic effect on the percentage of monocytes ingesting such a particle. The monocyte receptor essential for ingestion of desialated sheep erythrocytes or desialated sheep erythrocytes bearing C3b was inactivated by concentrations of trypsin that also prevented the monocytes from ingesting natural activators of the human alternative complement pathway, but did not alter the receptors for C3b or the Fc portion of IgG. The capacity of the nonimmune host to respond to desialated particles by initiating the monocyte ingestive process and by activating the alternative complement pathway to provide the synergy afforded by C3b deposition on that particle represents a primitive biochemical basis for differentiation of nonself from self.

A high-molecular-weight fragment of C3 was isolated from normal human serum by column chromatography, was generated by incubation of serum at 37 degrees C with inulin, and was produced from highly purified C3 by limited digestion with trypsin. This product was shown to inhibit the antibacterial function of neutrophils by using Escherichia coli O75 as the main test organism. The inhibitor reacted with anti-C3b and anti-C3c, but not with anti-C3B (anti-native C3) or anti-C3a. The manner of preparation of the inhibitor, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern, and the amino acid composition of the inhibitor indicated that it was fluid phase C3b. The inhibitor of neutrophil function (fluid phase C3b) was shown to bind to C3b receptors or acceptors on sheep erythrocytes in a model system.