We have investigated the effect of plasma fibronectin (Fn) on binding and phagocytosis of sheep erythrocytes (E) by human peripheral blood monocytes. Unopsonized E were not phagocytosed in the absence or presence of Fn, but Fn enhanced the phagocytosis of E bearing IgG. Sheep erythrocytes sensitized with IgM and C3b were ingested only when monocytes were exposed to Fn. The Fn enhancement of phagocytosis occurred for both fluid-phase and glass-adherent monocytes. Experiments in which Fn was washed out before mixing monocytes with opsonized E demonstrated that the Fn effect occurred because of interaction with the monocytes and not the opsonized particles. Chromatography of the Fn on Biogel A 1.5m showed that the phagocytosis-enhancing activity exactly co-chromatographed with the Fn protein. Fn did not increase the number of monocyte membrane receptors for the Fc fragment of monomeric IgG. We conclude that Fn enhances monocyte phagocytosis, not by binding to particles as a conventional opsonin, but by stimulating monocytes to ingest already opsonized particles more avidly.

We have investigated the interactions between plasma fibronectin (Fn) and human peripheral blood phagocytic cells. As shown by studies of the binding of Fn-coated fluorescent microspheres (Fn-ms), both polymorphonuclear leukocytes (PMN) and monocytes had specific binding sites for Fn at the plasma membrane. However, as purified from blood, only monocytes were stimulated by Fn to become more actively phagocytic. This increase in phagocytosis was reflected by an Fn-induced increase in the ingestion of IgG-coated erythrocytes and, more dramatically by an Fn-dependent initiation of phagocytosis of C3b-coated erythrocytes. Despite this difference between PMN and monocytes in the functional consequences of Fn binding, the cell surface molecules responsible for Fn binding on the two cell types shared many characteristics. On both cells, binding of Fn-ms was inhibited by sufficient concentrations of fluid-phase Fn; both PMN and monocytes bound fewer Fn-ms at 4 degrees C than at 37 degrees C; both achieved maximal binding at similar Fn-ms/cell ratios; and phenylmethylsulfonyl fluoride did not inhibit Fn-ms binding to either cell type. Most dramatically, monoclonal anti-Fn antibodies that inhibited binding of Fn-ms to one cell type inhibited binding to both; conversely, monoclonal anti-Fn antibodies that did not inhibit Fn-ms binding to either cell type did not inhibit binding to the other. Fn will stimulate PMN to a more actively phagocytic state, like that induced in monocytes, if the PMN are first exposed to C5a or N-formyl-methionyl-leucylphenylalanine. This effect occurs without apparent change in the number of Fn receptors. We conclude that the PMN and monocyte receptors for Fn are very similar, but that their milieu is very different in the two cells as purified from peripheral blood. Whereas Fn induces increased phagocytosis in monocytes, PMN must be activated before the Fn can be effective.

Plasma fibronectin (Fn) induces phagocytosis of C3b-opsonized sheep erythrocytes (EC3b) by human peripheral blood monocytes. However, Fn does not induce erythrophagocytosis of EC3b by human polymorphonuclear leukocytes (PMN), unless the PMN have been exposed to C5a or N-formyl- methionyl-leucyl-phenylalanine. Because of this difference, it is of great interest to examine Fn binding to cells that possess the capacity to differentiate into either granulocytes or monocytes. Hence, we have examined the consequences of Fn binding to the human myelomonocytic cell line, HL-60, both before and after in vitro differentiation of the HL-60, along a monocytoid or a granulocytoid pathway. Fn receptors were not found on undifferentiated HL-60, but several differentiating agents promoted the HL-60 binding of Fn-coated microspheres (Fn-ms). The peak of Fn-ms binding occurred four to five days after the induction of differentiation with dimethylsulfoxide (DMSO), and two days after induction by PMA. In addition, cells that differentiated along either the monocytoid or the granulocytoid pathway showed a marked increase in the phagocytosis of both IgG-coated erythrocytes (EA) and EC3b when they were exposed to Fn. Comparison of the effects of anti-Fn monoclonals on the binding of Fn-ms to the monocytes, PMN, and HL-60 showed that the same monoclonals block Fn-ms-binding and Fn-induced EC3b phagocytosis by all three cell types. Two monoclonal antibodies, M1/70 and A6F10, directed against membrane antigens on PMN and monocytes, inhibited Fn-ms binding. Both also blocked Fn-induced EC3b ingestion by these cells. However, neither antibody blocked Fn-ms binding or EC3b ingestion by differentiated HL-60. We conclude that differentiated HL-60 cells express functionally active Fn receptors, similar to monocytes and activated PMN, which, nonetheless, differ from normal cells in their association with the antigens recognized by M1/70 and A6F10.

A major problem in the cryopreservation of human polymorphonuclear leukocytes (PMN) is the loss of phagocytic function in cryopreserved cells. This is not a problem with cryopreserved monocytes. To study the reasons for this difference in detail, PMN and monocytes were either osmotically stressed in hypertonic media or were frozen to various temperatures. Cells were then returned to conditions of physiologic osmolarity and temperature. All cells remained viable. However, the ability of PMN to phagocytize bacteria and to bind sheep erythrocytes (E) opsonized with IgG, C3b, or C3bi decreased sharply after exposure to media of 600 mOsM or greater and after freezing to -1.5 degrees C. In contrast, monocytes were unaffected until a concentration of 1500 mOsM or a freezing temperature of -5 degrees C was exceeded. To determine whether the functional losses of surface receptor activity in PMN resulted from a loss of receptors from the membranes or from inactivation or internalization of receptors, opsonized E were incubated in the supernatants from stressed PMN. On subsequent incubation with healthy PMN, these E made fewer rosettes than control opsonized E. The inhibitory effect of the supernatants on rosetting of IgG-sensitized E could be removed by preincubation with IgG bound to Sepharose 4B. Immunoprecipitation of C3b and C3bi receptors from surface-iodinated, osmotically stressed, and control PMN suggested that about 50% of cell surface complement receptors were lost from the cell surface during osmotic stress. These experiments suggest that receptors for IgG and C3 are extruded from PMN cell membranes as a result of hyperosmotic stress, which is associated with the freeze-thaw cycle. This may be an early event in the functional damage done to PMN during attempts at cryopreservation.