By using antibodies against C5, C6, C7, C8, and C9, we found that terminal complement components were deposited on IgM-coated sheep erythrocytes (EIgM) kept in serum-free endotoxin-stimulated monocyte cultures for 24 or 48 h. Monoclonal antibodies revealed C9 neoantigens on the EIgM. There was no specific binding of an anti-S protein antibody, which reacts with the SC5b-9 complex, to the EIgM. Controls were native sheep erythrocytes (E) treated similarly which, in contrast to EIgM, do not activate the classical pathway of complement. Cycloheximide (1.0 microgram/ml) in the cell cultures resulted in no specific binding of the anti-C9 antibodies to EIgM. A fraction of the EIgM was lysed during incubation with the monocytes. We conclude that the monocytes secrete C5, C6, C7, C8, and C9, which form the membrane attack complex of complement (C5b-9) on the EIgM.

Binding of labelled protein to EIgM kept with macrophage or monocyte cultures with 3H-leucine under serum-free conditions, shows that de novo synthesis of protein with affinity to EIgM takes place. We find that monoclonal anti-C3c and anti-C3g antibodies and polyclonal anti-C4 and anti-C5 antibodies bind to such erythrocytes. This demonstrates that C4b, C3b and iC3b are deposited on the EIgM. Additional evidence for complement synthesis is the increase in binding of anti-C4 antibodies to EIgM when the incubation time was increased from 48 to 96 hours. Stimulation of the mononuclear phagocyte cultures with ET was necessary to obtain significant amounts of erythrocyte-bound complement proteins. From these results we conclude that the functional classical pathway of complement is produced in vitro by the monocytes and macrophages.

Monocytes cultured under serum‐free conditions secreted protein which bound covalently and non‐covalently to agarose beads, an activator of the alternative pathway of complement. There was a significant binding of monoclonal anti‐C3c antibodies, polyclonal anti‐C5, anti‐C6, anti‐C7, anti‐C8, and anti‐C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b‐9 or the membrane attack of complement (C5b‐9) is formed on the beads will be examined.

We studied the phagocytosis of agarose beads by human alveolar macrophages in terms of the morphology, the receptors involved, and the cellular substrates (plastic or fibronectin) used. Beads coated with C3b (58%) and iC3b (42%) by treatment with serum, were ingested during 45 min by CR1 and CR3 on the macrophages. This ingestion was inhibited 80-90% by the presence of polyclonal F(ab')2 anti-C3 fragments. Since the phagocytosis of both C3b- and iC3b-coated beads was about threefold stronger than for C3b-coated beads (trypsinized serum-treated beads), the results indicate that the CR3 is more phagocytic than the CR1. The phagocytosis of initially complement uncoated beads, which are slowly opsonized with macrophage-produced C3b and iC3b in vitro, was also strongly inhibited (70-80%) by the presence of anti-human C3 F(ab')2 fragments. There was an increased phagocytosis (10-17%) of complement precoated beads by macrophages cultured on the fibronectin substrate versus the plastic substrate. The morphology and rapid phagocytosis of the complement precoated beads was demonstrated by SEM. The general impression was that membranous protrusions stretched towards the beads, which became increasingly enclosed by plasma membrane.

Attachment of protein to agarose beads cultured with macrophages in protein-free medium containing 3H-leucine, shows that de novo synthesis of protein with affinity to the beads takes place. We also found that monoclonal antibodies against human C3c, C3g, and a C9-neoantigen as well as polyclonal antibodies against human C5 and C9, bound to agarose beads that had been kept with the macrophage cultures. Demonstration of C3 derivatives on the agarose beads shows that the essential complement factors of the alternative pathway are synthesized and have been activated by the beads. Deposition of C5 and the detection of a neoantigen of C9 on the beads, indicates that the whole terminal complement pathway has been formed and activated. We conclude that human alveolar macrophages form in vitro the functional alternative pathway of complement, C5 and C9, and we have indirect evidence for synthesis of C6, C7, and C8.

We have studied the binding of fibronectin to complement (C3b, C3bi, C3d)‐coated agarose beads and its effect on cell association of such beads to mouse macrophages. Fibronectin bound to agarose beads preincubated in human serum, whereas no binding occurred after preincubation of the beads with complement‐inactivated (50°C for 20 min or ethylenediaminetetraacetic acid) sera. The binding of iodine‐labelled fibronectin to beads preincubated in fibronectin‐depleted serum (HS‐FIB) was about twice that of beads preincubated in normal serum. Unlabelled fibronectin inhibited the following binding of labelled fibronectin to beads pretreated in HS‐FIB. A similar amount of fibronectin bound to agarose beads coated with equimolar amounts of C3b, C3bi, or C3d, suggesting that the common domain C3d carries the main binding site(s) for fibronectin. Preincubation of serum‐treated and trypsinized agarose beads with fibronectin led to an increased association (22%) of such beads to mouse macrophages. The results indicate that fibronectin promotes binding of complement‐coated agarose beads to mouse macrophages, whereas the ingestion of the beads is mediated via complement C3 receptors.

Mouse peritoneal macrophages stimulated with insoluble glycans in vitro release high amounts of acid hydrolases, N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase, and beta-D-galactosidase. The most potent of the stimulatory glycans is a beta-1,3-D-glucan isolated from yeast cell walls. Up to 50% of total enzyme activity was found in the medium after stimulation with this glycan for three days. Agarose, another insoluble glycan containing an alternating sequence of the disaccharide beta-1,3-D-galactose-alpha-1,4-3,6-anhydro-L-galactose units was less potent. The soluble beta-1,3-D-glucan laminaran, which also contains small amounts of mannitol, was not able to induce release of acid glycosidases from macrophages. The release was independent of serum since macrophages cultured under serum-free conditions showed nearly the same pattern of enzyme activities, both in the cells and media. There was no increased release of the acid hydrolase alpha-D-mannosidase after stimulation with the insoluble beta-1,3-D-glucan for three days. The release of the lysosomal glycosidases was not due to cell death, since only small amounts of the cytoplasmic enzyme lactate dehydrogenase were found in the culture media. Insoluble polystyrene latex particles were not able to stimulate mouse macrophages to release lysosomal glycosidases. Tritiated glycans (amylose, dextran, laminaran, the insoluble beta-1,3-D-glucan, and agarose) and the p-nitrophenyl-glycopyranoside derivatives were used as substrates to investigate whether the macrophages contained or released glucanases capable of degrading alpha-1,4-D-glucans, alpha-1-6-D-glucans, beta-1,3-D-glucans, and agarose respectively. We conclude that the glycans were not degraded in macrophage cultures during the time period tested nor were the enzymes induced in macrophages by the glycans during in vitro culture for seven days.

We have examined to what extent human fibronectin associated with agarose beads with a 5- to 10-micron diameter mediates binding and uptake of the beads by mouse macrophages and human monocytes. Native agarose beads preincubated with 125I-fibronectin were neither associated with nor taken up by mouse macrophages after 30 min of incubation under serum-free conditions. When fibronectin was cross-linked to cyanogen bromide-activated agarose beads or incubated with gelatinized beads, this resulted in a significant increase in particle binding by macrophages and monocytes as compared with gelatinized beads, whereas the fraction of cells with ingested particles remained unaltered. Native agarose beads activated by cyanogen bromide and treated with ethanolamine were to a greater extent associated with and taken up by phagocytes than fibronectin- or gelatin-coated beads. Our results indicate that fibronectin acts as an adhesive glycoprotein and not as an opsonin. Since agarose beads are activators of the alternative pathway of complement, and fibronectin is reported to bind to factor C3, we speculate that cell-derived C3b is bound to the beads and fibronectin-coated beads are ingested by the phagocytes via complement C3b receptors on the cells.

Agarose beads (diameter 5–10 μm) preincubated in human serum became associated (attached and ingested) to 50–60% of the salmon macrophages within 60 minutes. However, Beads preincubated in serum treated with heating (50°C, 20 min) or with EDTA (10 mM) to inhibit the activation of alternative complement pathway, were not associated to the phagocytes. Furthermore, agarose beads coated with human C3b and C3bi after incubation with isolated complement factors (C3, D, B), were associated to 30–40% of the phagocytes. About 80% of the cell-associated agarose beads was intracellularly located. Conversion by trypsin treatment (0.01%) of agarose bound C3bi to C3d, abolished the association of such beads to the macrophages. The results demonstrate that salmon macrophages posess complement receptors that bind human C3b and C3bi. Agarose beads coated with these ligands (C3b and C3bi) are attached and ingested by the phagocytes.

The phagocytosis of particles activating the alternative pathway of complement by human monocytcs cultured under serum‐free conditions was studied. In contrast to native zymosan particles, which were easily ingested, rabbit erythrocytes and agarose heads had to be coated with C3b or C3bi to be engulfed by the monocytes. The binding and ingestion by monocytes of particles coated with C3bi were greater than for the same particles coated with the equivalent amount of C3b. The binding and uptake of rabbit erythrocytes and agarose beads were proportional to the amount of C3b or C3bi on the particles. In contrast to the complement activator particles, C3b‐ and C3bi‐coated sheep erythrocytes, which are non‐activators, were not ingested by the monocytes, although attachment to the monocytes took place. The presence of methylamine or cobra venom factor, which are complement inhibitors, strongly reduced the ingestion of native zymosan by the monocytes, whereas the uptake of C3b‐ or C3bi‐coated zymosan particles were only weakly affected. This suggests that the binding of native zymosan to monocytes is sensitive to interference from a cell‐derived alternative pathway C3 convertase (C3bBb). Binding and uptake of activators by human monocytes via complement receptor(s) are discussed.

We have determined the receptors on human monocytes and mouse peritoneal macrophages producing agarose binding. By using isolated human complement factors C3, B and D, agarose beads were coated with C3b. In some experiments C3b was converted to C3bi by using human serum diluted 1:20. Agarose beads coated with C3b or C3bi bound strongly to monocytes. Only agarose beads coated with C3bi were attached to mouse macrophages. Trypsinization of agarose beads coated with C3bi abolished the attachment of the beads to macrophages and monocytes, probably because of conversion of C3bi to C3d. Endocytosis by macrophages of agarose preincubated in human serum or in C5‐deficient AKR mouse serum reached the same levels, indicating that the amount of C5 present in serum during preincubation is not important for the degree of endocytosis. It is concluded that internalization of agarose by macrophages is mediated via the C3bi receptor.

The phagocytosis by macrophages of C3bi‐coated agarose heads reached a plateau after 15 min, compared with 30 min for C3b‐coated beads. By using 125I‐Iabelled C3bi or C3b coupled to the agarose beads, we found that 70% and 95% of total radioactivity were removed from the heads after 12 h and 36 h of intracellular digestion, respectively. Intracellular degradation of C3bi linked to agarose beads was also demonstrated by testing binding of monoclonal antibodies against human C3c, C3g and C3d to beads extracted from the cells after phagocytosis. Such extracted beads also showed reduced attachment to new macrophages compared with non‐ingested beads. Treatment of the cells with leupeptin, an inhibitor of the lysosomal enzyme cathepsin B, or with dextran sulphate to inhibit phagosome‐lysosome fusion greatly reduced the release of labelled protein from the agarose during the first 12 h. These findings show that C3bi and C3b on agarose is destroyed intracellularly by lysosomal enzymes.

Phagocytosis of agarose beads by macrophages. cultured under serum‐free conditions was studied. 48 h was needed before a plateau in the uptake was reached. The ingested agarose beads were coated extracellularly with macrophage‐derived protein before attachment and ingestion of the beads. Intracellularly, the agarose‐linked protein was removed from the agarose. If the ingested agarose beads were extracted from the macrophages within 24 h after the plateau in the uptake was reached, a fraction of the beads could attach to new macrophages, demonstrating modification of the agarose beads by opsonin(s). Because of binding of anti‐human C3c antibodies to beads extracted from the macrophages after 24 h of phagocytosis and the trypsin sensitivity of the protein on the agarose, we conclude that the main opsonin on the agarose beads is C3bi. Requirements for the stimulatory effect of agarose on macrophages are summarized.

Macrophages stimulated by an insoluble beta-1,3-D-glucan from yeast cell walls were able to destroy tumour cells as measured by the release of radioactive label from prelabelled 14C-thymidine cells. Target cells were B-16 melanoma, P-815 mastocytoma, and the L-929 cell line. A significant target cell killing by macrophages stimulated by glucan was observed after 72-96 h. The cytolysis of L-929 cells was investigated in some detail. No stable soluble cytolytic factor appeared to be released into the medium during the stimulation of macrophages by glucan, since cell-free spent medium had no cytotoxic effect on L-929 cells. The densities of the macrophage monolayers were critical for an effective target cell killing; dense cultures showed more cytotoxicity than less dense cultures. The kinetics of the development of macrophage-mediated cytotoxicity suggests a minimum stimulation period of 4 days for maximal cytolysis.

Agarose stimulation of macrophages in vitro was studied. Under conditions where agarose was ingested, stimulation was detected during 24–48 h of incubation at a time when the agarose increasingly was concentrated in the perinuclear region. Removal of extracellular agarose after 24 h when endocytosis had reached a plateau did not reduce the stimulatory effect. Preincubation for 4 days with dextran sulphate in concentrations reported to inhibit phagosome‐lysosome fusion potentiated strongly the stimulatory effect. In all situations in which agarose was not internalized—in teflon tubes where the cells remain in suspension, on glass cover slips with inhibitors (2‐deoxy‐D‐glucose, cytochalasin B), or with large, noningestible Sepharose beads—no stimulation was recorded. The possibility is discussed that stimulation of macrophages by agarose may be related to complement activation in phagosomes.

We have studied the endocytosis of tritium-labelled and of non-radioactive agarose in mouse macrophages in vitro. The endocytosis was greatest and most rapid in syngeneic mouse serum and in human serum, reaching a plateau after 12 h of incubation. Ten per cent serum was the minimum concentration giving optimal ingestion. The endocytosis appeared to be regulated by mechanisms involving complement factors C3 and B. Different pretreatments of sera, inactivating or depleting C3 and B, resulted in 70-80% reduction of endocytosis. Preincubation of agarose in untreated serum increased the endocytosis of agarose in heat-inactivated serum three-fold indicating that the essential factors were bound to agarose. Antibodies against C3 and B reduced endocytosis moderately but significantly.