Increased expression of C3b receptors on polymorphonuclear leukocytes induced by chemotactic factors and by purification procedures.

Abstract

The expression of C3b receptors on the surface of peripheral blood polymorphonuclear leukocytes (PMN), monocytes, and lymphocytes was determined by measuring the immunofluorescence of these cells after they had been indirectly stained with F(ab')2 anti-C3b receptor. The assay employed flow cytofluorography and yielded estimates of relative numbers of cellular C3b receptors that correlated (r = 0.99) with the measurement of these sites by the binding of 125l-dimeric C3b. Incubation for 30 min at 37°C of washed buffy coat cells that had been prepared at 4°C by centrifugation of fresh, citrated blood increased by as much as eightfold the availability of C3b receptors on PMN and monocytes when compared with the limited expression of receptors on these cells in buffy coat preparations held at 0°C or on cells in whole citrated blood held at 37°C for 30 min. The expression of additional antigenically detectable C3b receptors on PMN and monocytes in the washed buffy coat preparations occurred by 15 min incubation at 37°C and was impaired when cells were incubated at temperatures ≤28°C. Quantitating the number of functional receptors by uptake of 125l-dimeric C3b indicated that purified PMN that had been prepared at 4°C and at 20°C had 5500 C3b receptors per cell and 21,000 C3b receptors per cell, respectively; incubation of both preparations of purified PMN for 30 min at 37°C induced the expression of 38,000 C3b receptors per cell. The sub-population of lymphocytes bearing C3b receptors in buffy coat preparations held at 0°C differed from PMN and monocytes in having fourfold to fivefold more available receptors and in exhibiting only slight increases in receptor expression after cells had been incubated at 37°C. PMN in citrated whole blood held at 37°C and not subjected to centrifugation did not increase their expression of C3b receptors. However, addition to blood of purified C5a desArg or of f-Met-Leu-Phe in nanomolar concentrations caused dose-related increases of as much as eightfold to 10-fold in receptor expression on PMN. The chemotactic peptides did not further augment the availability of C3b receptors on PMN in washed buffy coat preparations incubated at 37°C. The number of receptors for f-Met-Leu-[3H]Phe on PMN purified at 4°C was 14,670 per cell and 11,840 per cell, and increased to 41,690 per cell and 29,350 per cell, respectively, on PMN purified from replicate samples of blood at 20°C. Similar increases in the number of receptors for 125l-C5a desArg on PMN did not occur, because cells purified at 4°C had 53,350 receptors per cell and 42,890 receptors per cell, and cells purified from replicate samples of blood at 20°C had 53,040 receptors per cell and 48,000 receptors per cell, respectively. Thus, at least two receptors on PMN, those for C3b and for f-Met-Leu-Phe, reside in expressed and latent forms, whereas the unstimulated PMN apparently fully expresses its content of C5a desArg receptors.