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A subpopulation of human B lymphocytes forms rosettes with mouse erythrocytes through a glycoprotein-dependent bond. Further studies of this bond show that the lymphocyte receptor is not immunoglobulin, although the binding of antisera or staphylococci to surface immunoglobulin inhibits the formation of mouse rosettes. Rosette formation could not be induced in thymocytes by enzymatic modification of the surface, or in T cells by lectin-induced transformation. The capacity to bind mouse erythrocytes was lost after incubation in a serum-free medium of lymphcytes from most patients with chronic lymphocytic leukaemia (CLL) and normal subjects. This loss could be prevented by the addition of a variety of sera and glycoprotein-containing substances to the medium, including fetuin. Conditions conductive to the subsequent restoration of rosetting capacity could not be found, indicating that the loss of this capacity was not due to the shedding of a cell-surface receptor which could be re-synthesized. It is suggested that the functional receptors in B1 lymphocytes are held in aggregates by cross-linking peripheral glycoprotein molecules; disaggregation and consequent loss of the capacity to form rosettes with mouse erythrocytes occurs during incubation in serum-free media, and during maturation of lymphocytes to the B2 stage.

The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation in lymphocytes from patients with chronic lymphocytic leukaemia (CLL). The differentiation was detected by the appearance of cytoplasmic immunoglobulin (CIg) and a plasmacytoid morphology, and was accompanied by the loss of the ability of the CLL cells to rosette with mouse erythrocytes (M). Loss of M rosetting occurred rapidly (within 10 min), was not prevented by cycloheximide or actinomycin D and was elicited by low concentrations of TPA (ED50 8 X 10(-10) M). Inhibition of rosette formation was induced by other phorbol diester promoters, but not non-promoting derivatives. Loss of the capacity to form M rosettes occurs before events which are evidently related to genome activation.

The calcium ionophore A23187 acted synergistically with phorbol dibutyrate (PDBu) to stimulate human neutrophil superoxide production. A23187 shortened the lag period and markedly increased the initial rate of neutrophil superoxide production induced by suboptimal concentrations of PDBu. 1 microM A23187 reduced the EC50 value for superoxide release from 56 to 8 nM PDBu. This effect of A23187 was correlated with enhanced binding of [3H]PDBu to its receptor and a reduction in the dissociation constant (Kd) from 27 to 10 nM, without altering the apparent total number of phorbol dibutyrate receptors. These actions of A23187 were abolished in the presence of EGTA or TMB-8, confirming a dependence on Ca2+.

Using rosetting tests with untreated mouse erythrocytes (M) and pronase-treated M (pro M), four human B cell subsets can be identified. Three of these, possessing the phenotypes BM+ pro M+, BM- pro M+ or BM- pro M-, constitute 17%, 61% and 22% of normal blood B cells respectively. The fourth subset, BM+ pro M-, does not occur in normal tissues but was found in the pre-B-cell line of Raji cells, indicating that this phenotype may be a marker for early B cells. Some differences in the proportion of each subset were found in cord blood, lymph nodes and tonsils. Surface-immunoglobulin-positive (SIg+) and -negative (SIg-) non-T cells were present in each subset. M and pro-M rosetting tests were applied to cells from blood of 27 cases of chronic lymphocytic leukaemia (CLL) and to cells from involved nodes, spleen or marrow in five cases of non-Hodgkin's lymphoma (NHL). In 15 cases of CLL, there was considerable increase in the BM+ pro M+ subset (BM+ pro M+ type CLL); in seven cases, there was a predominance of BM- pro M+ cells and in another four cases, BM- pro M- cells predominated. All five cases of NHL were greatly enriched in BM- pro M- cells. There was no obvious correlation between rosetting and other surface markers but BM- pro M- clones in CLL or NHL always stained brightly with FITC-anti-Ig. This was not found in BM+ pro M+ or BM- pro M+ clones. Rosette formation of neuraminidase-treated B cells with M identifies the same subset as B-pro-M rosetting in normals and CLL. Evidence is presented that two types of receptors are involved in M and pro-M rosetting, designated R1 and R2, binding to corresponding M ligands L1 and L2. M rosetting is due to R1-L1 binding while R2-L2 binding mediates B-pro-M rosetting. Shifts between subsets within the same clone in some cases of CLL suggest that the subsets are distinct maturational stage of B-cell development rather than families of B cells of different lineage. The following B-cell maturation sequence is proposed: R1+ R2- lead to R1+ R2+ leads to R1- R2+ leads to R1- R2-.

Previous studies have shown that auranofin (AF), a lipophilic gold I complex, modulates metabolic events in leukocytes stimulated by phorbol esters, whose major cellular binding site is now known to be the Ca++/phospholipid-dependent protein kinase (protein kinase C). In these experiments we have investigated the effect of AF on the binding of phorbol dibutyrate (PDBu) to human chronic lymphocytic leukemia (CLL) B cells. AF enhanced binding of PDBu to its receptor in CLL cells by a) causing an increase in the affinity of PDBu receptors from Kd 20.3 nM to 7.3 nM, and b) enhancing translocation of PDBu receptors to the cell membrane. The increase in PDBu binding induced by AF in whole cells was only partially reversible by EGTA or the intracellular Ca++ antagonist TMB-8. Studies performed with quin-2-labeled cells showed that 100 microM AF caused a mean (+/- SD) rise in cytosolic Ca++ levels from 0.41 (0.12) to 0.85 (0.33) (n = 5). Thus the mechanism by which AF increases binding of PDBu to its receptor appears to be partially dependent on Ca++. These effects of AF occurred at cellular levels achieved in mononuclear cells during chrysotherapy of patients with rheumatoid arthritis.

The calcium ionophore A23187 synergised with phorbol dibutyrate-induced activation of human chronic lymphatic leukaemia B-cells, as assessed by modulation of the membrane receptor for mouse erythrocytes. Thus A23187 (1 microM), which alone had no effect on expression of the receptor for mouse erythrocytes, reduced the EC50 and shortened the lag period for modulation of this receptor by phorbol dibutyrate. This action of A23187 was shown to be due to enhanced binding of [3H]phorbol dibutyrate to its receptor (phospholipid/Ca++ dependent protein kinase C) whose affinity was altered from a predominantly low affinity state (Kd 83 nM) to high affinity (Kd 9 nM). A23187 had no effect on the total number of phorbol dibutyrate receptors. EDTA abolished these actions of A23187.