Characterization of surface receptors on bovine leukocytes

Three bovine leukocyte populations--peripheral blood lymphocytes (PBL), mammary gland polymorphonuclear neutrophils (PMN) and macrophages (Mo)--were characterized with respect to five surface markers: surface immunoglobulin (SIg), sheep erythrocyte receptor, complement (C) receptor and Fc receptors for both IgG and IgM. The majority of PMN and Mo possessed C and Fc receptors for IgG, but lacked SIg and the erythrocyte receptor. The PMN, but not Mo, also expressed a Fc receptor for IgM. The PBL were heterogeneous with respect to their surface characteristics and evidence was presented for the following subtypes: (a) cells with the E receptor alone; (b) cells with E receptor plus the Fc(IgG) receptor; (c) cells with SIg plus the C receptor but minus the Fc(IgG) receptor; (d) lymphocytes with SIg plus the C receptor and the Fc(IgG) receptor, and (e) cells lacking E receptors and SIg but bearing Fc(IgG). It was assumed, but not proven, that some of these latter cells must also bear the C receptor. The significance of the various cell types in antiviral defense is briefly discussed.     

Independent movement of surface immunoglobulin from Fc receptors on lymphocyte membranes.

The majority of surface immunoglobulin-positive lymph node cells possess Fc receptors detectable by a rosette technique. The movement of surface immunoglobulin to form caps does not alter the distribution of Fc receptors, although Fc rosette-forming indicator cells collect over the immunoglobulin cap under capping conditions.     

Surface receptors on human haematopoietic cell lines.

The expression of complement receptors, of Fc receptors, of SRBC receptors and of S-Ig was investigated on human haematopoietic cell lines of proved malignant derivation. According to their origin and to a panel of phenotypic markers these lines have been classified into lymphoma lines, myeloma lines and leukemia lines. Results were compared with those obtained on non-malignant EBV carrying lymphoblastoid cell lines (LCL). Among the lymphoid cell lines the LCL showed a pattern of B-lymphocyte surface markers, i.e. surface immunoglobulins, C3 receptors but low density of Fc receptors. The non-Burkitt lymphoma lines bore in varying degree these B-lymphocyte markers. The lines U-698 M and DG-75 were exceptional in having only surface immunoglobulin. The Burkitt lymphoma lines had all B-lymphocyte markers. The myeloma lines differed from the lymphoid lines in lacking C3 and Fc receptors and showed only trace amounts of surface immunoglobulins. In contrast to lymphoid and myeloma lines, the leukaemia lines were completely lacking surface immunoglobulins, but showed C3 and Fc receptors in variable densities. On line, the ALL derived line MOLT-3 showed the capacity to spontaneous rosette formation with SRBC. The findings that LCL presented a homogeneous pattern of B-lymphocyte surface markers may be of value in order to discriminate between these lines and lines derived from haematopoietic malignancies other than Burkitt lymphomas.     

Surface markers on lymphocytes of multiple sclerosis patients.

Peripheral blood lymphocytes of twenty-two multiple sclerosis (MS) patients and thirty-five healthy controls were examined for the presence of surface markers characteristic for B lymphocytes (surface immunoglobulin, receptor for C3 (EAC), reporter for Fc (EA) and the spontaneous rosette-forming capacity characteristic of T cells. The results obtained indicate that the number of B and T cells in MS is similar to controls, as evaluated by the presence of surface immunoglobulin and E rosette-forming capacity. However, a statistically significant reduction in the percentage of lymphocytes bearing C3 receptors has been found in MS patients. It might have resulted from a reduction in the lymphocyte population bearing C3 receptor but no surface immunoglobulin. The EA rosette test revealed the greatest difference between the groups. The difference indicated a reduction in the density of the receptor for 7S Fc on lymphocytes from MS patients. The results obtained are consistent with the hypothesis of an immune deficit in multiple sclerosis.     

Human peripheral lymphocytes bearing surface immunoglobulin do not have readily detectable Fc receptors.

The question of whether human peripheral B lymphocytes have Fc receptors was examined directly by double-label immunofluorescent techniques utilizing assays for detection of Fc receptors, surface immunoglobulin, and complement receptors. Fc receptors were detected by indirect immunofluorescence after incubation with soluble antigen-antibody complexes. Complement receptors were detected by the binding of fluoresceinated bacteria coated with complement. It was demonstrated that most surface immunoglobulin-bearing, complement-receptor positive lymphocytes did not bind soluble antigen-antibody complexes. Conversely, most cells which readily bound soluble complexes did not have surface immunoglobulin or complement receptors. Therefore, most peripheral B lymphocytes do not have easily detectable Fc receptors and most Fc receptor-bearing lymphocytes do not have B cell markers.     

Relationship of chemotactic receptors for formyl peptide and C5a to CR1, CR3, and Fc receptors on human neutrophils

The co-expression of C5a and formyl-methionine-leucine-phenylalanine-lysine (FMLPL) receptors with CR1, CR3, and Fc receptors on human neutrophils (PMN) was studied. Fluorescein-conjugated C5a (FL-C5a) and FMLPL (FL-FMLPL) were used to identify C5a and formyl peptide receptors. CR1, CR3, and Fc receptors were identified with monoclonal antibodies and a Texas red-labeled goat anti-mouse immunoglobulin second step reagent. The co-expression of chemotactic receptors with CR1, CR3, or Fc receptors was evaluated using two-color flow cytometry. A direct correlation between the degree of expression of receptors for FL-FMLPL and the expression of CR3, CR1, and Fc receptors on individual PMN was observed. In contrast, no correlation between the degree of C5a receptor expression and CR1, CR3, or Fc receptor expression was found. Similar results were obtained with PMN after up regulation of CR1, CR3, Fc, and FMLPL receptors by incubation at 37 degrees C for 10 min with or without phorbol myristate acetate. These data suggest that the expression of FMLPL, CR1, CR3, and Fc receptors are regulated in a similar manner, whereas C5a receptor expression is regulated independently. Furthermore, these data indicate that within a given population of PMN, a parallel exists between the degree of CR1, CR3, FMLPL, and Fc receptor expression on individual cells.     

Effects of interleukin-3 with or without the c-kit ligand, stem cell factor, on the survival and cytoplasmic granule formation of mouse basophils and mast cells in vitro.

We assessed the ultrastructure and the cell-surface expression of receptors for immunoglobulin E (Fc epsilon R), and c-kit, the receptor for stem cell factor (SCF), in mouse basophils and mast cells present in short-term cultures of mouse bone marrow cells in interleukin-3 (IL-3) with or without SCF. Basophils did not develop increased numbers of cytoplasmic granules and underwent apoptosis in cultures containing IL-3 and SCF, whereas mast cells thrived and developed increased numbers of granules. Basophils were nearly all Fc epsilon R+ c-kit- when sorted after culture in IL-3 and SCF; most mast cells were Fc epsilon R+ c-kit+. However, a second population of Fc epsilon R+ c-kit- mast cells was present after culture in IL-3 and SCF. These c-kit receptor-negative mast cells were less mature than c-kit+ mast cells and contained significantly fewer cytoplasmic granules than the c-kit+ mast cells present in the same cultures (P < 0.001). Thus, mouse basophils express little or no c-kit receptor on their surface, nor can they survive for long periods in SCF-supplemented cultures. By contrast, mouse mast cells seem to express the Fc epsilon R early in their development, even before they express detectable c-kit receptors on their surface. IL-3 promotes cytoplasmic granule formation in immature mast cells, but even more granules are formed when c-kit receptor-positive immature mast cells are cultured in both SCF and IL-3.     

Isolation of various canine leucocytes and their characterization by surface marker analysis.

Various techniques were used to separate canine peripheral blood leucocytes into populations enriched in lymphocytes, polymorphonuclear leucocytes, phagocytic mononuclear cells (monocytes) and macrophages. Surface markers on each cell population were determined by rosette formation. Fc receptors for IgG and complement receptors (C3b and C3d) were present on PMN, monocytes, macrophages as well as on a sub-population of lymphocytes. Purification of the lymphocytes into T-and B-cell-enriched populations revealed that these receptors were present only on the B lymphocytes and not on the T lymphocytes. In addition, a third lymphocyte population, which did not possess surface immunoglobulin, and Fc receptor but not the complement receptor. None of the cell populations exhibited C4 complement receptors or Fc receptors for IgM. When different cell populations were tested for their ability to form rosettes directly with human type 'O' red blood cells it was found that most populations could rosette, suggesting that this technique could not be used as a specific marker for canine T lymphocytes.     

Surface Markers on Human B and T Lymphocytes

The distribution of surface immunoglobulin and receptors for Fc, C3, and sheep erythrocytes (SRBC) on resting and blast-transformed peripheral lymphocytes was investigated. The following conclusions were reached. [1] SRBC receptors were retained on all blast-transformed T lymphocytes. No such receptors were found on normal or neuraminidase-treated B lymphocytes. [2] Receptors for Fc and C3 were found to be expressed on the same resting lymphocytes, which formed a subpopulation that did not entirely overlap with surface immunoglobulin-positive B cells. Owing to this and the fact that Fc/C3 receptors were lacking on almost all B blast cells, as well as for other reasons elaborated in the text, it is argued that caution must be taken when these receptors are used as B-cell markers. [3] A comparison among C3 indicator cells prepared with human or mouse complement showed that these detected the same lymphocyte subpopulatiom [4] B blast cells, induced by 72-hr pokeweed mitogen cultures, were found to carry detectable amounts of surface immunoglobulin. [5] Phagocytic leukocytes were found to be conveniently detected by scoring the number of cells with internal C3 indicator cells after osmotic lysis of externally bound indicator cells.     

Characterization of Human Null Cells Isolated from Peripheral Lymphocytes by a Simultaneous Double-Rosetting Procedure

Human null cells were isolated from peripheral blood lymphocytes (PBL) by differential centrifugation on a Ficoll-Hypaque gradient of the PBL following simultaneous rosetting with erythrocyte indicators specific for B and T lymphocytes. Specifically, the T lymphocytes were rosetted with 2-aminoethylisothiuronium bromide-treated sheep erythrocytes (ShE), whereas the B lymphocytes were either rosetted with ShE coated with xenoantibodies against human gamma globulin or first sensitized with monoclonal antibodies to human Ig-like antigens and then rosetted with ShE coated with xenoantibodies against mouse gamma globulin. Approximately 90% of the lymphocytes isolated were null cells that did not bear detectable B-cell markers-that is, surface immunoglobulin and/or Ia-like antigens-or T-cell markers-that is, ShE receptors. The large majority of the null cells expressed receptors for the IgG Fc fragment (53-93%), C3 component (65-92%) and monkey erythrocytes (60-91%) but lacked receptors for the IgM Fc fragment and murine erythrocytes. The null cells exhibited high natural killer cell activity and antibody-dependent cellular cytotoxicity and were two- to four-fold active than the total PBL and the T-enriched cell fractions. The null cells, however, did not respond to stimulation with phytohaemagglutinin and failed to function as either stimulators or responders in an unidirectional mixed lymphocyte reaction.     

Effects of surface-active agents on neutrophil receptors.

An easily performed assay to identify the C3b and Fc receptors on human neutrophils was developed. Salmonella typhimurium were treated with fluorescein and then incubated in nonimmune fresh human serum, which led to C3b fixation via activation of the alternative pathway. Similarly, type II pneumococci were treated with fluorescein and opsonized with type-specific rabbit antiserum. Neutrophils bearing C3b and Fc receptors formed rosettes with the respective bacteria, which were easily readable because of their bright fluorescence. Incubation of neutrophils at 37 degrees C with C3-coated bacteria generated 54 +/ 4% C3b rosettes, whereas neutrophils incubated with immunoglobulin G-coated bacteria yielded 75 +/ 7% rosettes. Incubation at 4 degrees C inhibited the formation of C3b rosettes but not Fc rosettes. Heat inactivation of the fresh human serum at 56 degrees C for 30 min completely inhibited the formation of the C3b rosettes, and addition of heat-aggregated immunoglobulin G to the polymorphonuclear leukocyte blocked the ability of the polymorphonuclear leukocyte to bind immunoglobulin G-coated bacteria. Addition of 1.0 mM N-ethylmaleimide, 0.1 mg of trypsin per ml, 10 mM H2O2, O2- generated by xanthine-xanthine oxidase, and 8 times 10(-4) M hydrocortisone inhibited the C3b receptor, but did not inhibit the Fc receptor. In neutrophils, the selective effect of the various inhibitors suggests that the Fc and C3b receptors are distinct entities.     

Subpopulations of Human Lymphoblastoid Cell Lines

Forty lymphoblast cell lines derived from normal subjects, patients with infectious mononucleosis, leukemia, and Burkitt's lymphoma have been studied for surface receptors including surface Ig, complement receptors by the EAC rosette and fluorescent (Raji cell) techniques, and Fc (aggregate) receptor by direct and indirect immunofluorescence. Because of the B-cell tropism of the Epstein-Barr virus (EBV), an effort was made to correlate the expresion of various surface properties of lymphoblastoid cell lines with the content of EBV viral DNA as determined by complementary RNA-DNA (cNRA-DNA) hybridization on membrane filters or by DNA-DNA renaturation kinetic analysis. The only correlation established was with the Fc receptor determined by direct immunofluorescence. No correlation of EBV genome equivalents per cell with complement receptor or surface Ig was noted, suggesting that the expression of these receptors is not influenced by EBV viral DNA content. Subgroups of lymphoblastoid cell lines were on the basis of variable expression of surface receptors, designated B1, B2, B3, B4, and T. The distribution of lymphoblastoid cell lines into these subgroups were in the ratio of 14:4:1:4:1. The B1, B2, and B4 cell lines (except Molt 4F) were found to contain EBV. The B3 subgroup, for wich cell line 698 was the sole example, expressed surface immunoglobulins but no other B-cell characteristics, and H.S.B., a T-cell line, lacked detectable EBV.     

Relationship of mouse erythrocyte binding receptor on peripheral blood mononuclear cells to receptors for IgG Fc and C3

When peripheral blood mononuclear cells separated at 4 degrees C were incubated at 37 degrees C, they shed their surface receptors. Total absorption of the released mouse erythrocyte binding receptors from the supernatant of the cell suspension with mouse erythrocytes did not lead to a change in the IgG Fc and C3 receptor content of the supernatant. On removal of the C3 or IgG Fc receptors, the quantity of mouse erythrocyte binding receptor remained unchanged. These observations indicate that the three receptors are mutually independent structures, or undergo dissociation in the course of the shedding.     

Immunopathology of natural and experimental lymphomas induced by wild mouse leukemia virus

Naturally occurring lymphomas of Lake Casitas (LC) wild mice, and the lymphomas induced by LC murine leukemia virus (MuLV) in Swiss mice from the National Institutes of Health, displayed remarkably similar gross, microscopic, and functional characteristics. They spared the thymus, arose primarily in the splenic red pulp, became leukemic, and were comprised of stem cells lacking classic T- and B-cell markers. Cytoplasmic and surface immunoglobulin were undetectable in 34 of 35 spontaneous LC lymphomas and in any of ten LC MuLV-induced lymphomas in NIH Swiss mice. Assays for immunoglobulin secretion, complement (C'3) and Fc receptors, Thy 1.1,2 antigens, Ly 1,2 antigens, and erythroid and myeloid markers were negative on all of the spontaneous and experimental lymphomas. Cell lines were derived from five spontaneous lymphomas of LC mice. Three lines were characterized as null cells, one line as B cells, and one line as macrophages. All cell lines were diploid. The wild mouse spontaneous lymphomas, and lymphomas experimentally induced by LC MuLV in laboratory mice, provide a useful model for childhood acute lymphoblastic leukemia and for study of the early steps of B-lymphocyte differentiation.     

Immunoglobulin (Fc) receptors on murine T- and B-lymphocytes: investigations using tumor models

Lymphoid tumors are productive experimental models for the study of lymphocyte immunoglobulin receptors. Investigations with Fc receptor expressing lymphoid tumor cells have generated much useful information about: (a) the developmental expression of the different classes of Fc receptors on lymphoid cells of the T- and B-lineages; (b) the biochemical steps involved in the regulation of Fc receptor expression on lymphoid cells; (c) the structures of lymphoid cell Fc receptors and their genes; (d) the signals that induce alterations in the expression of Fc receptors on lymphoid cells; and (e) the molecular specificity of the binding of immunoglobulin to lymphoid cells Fc receptors. In addition, tumors that secrete immunoglobulins are providing useful models for analysis of the mechanisms by which B-cells influence Fc receptor expression and function on T-cells. An interesting, bi-directional immunoregulatory circuit involving Fc epsilon R+ host T-cells and IgE-secreting hybridoma cells has been identified that could prove useful in the analysis of the regulation of epsilon heavy chain expression. The studies discussed in this article and elsewhere in this volume serve to emphasize that, in addition to being clonal sources of key molecules such as Fc receptors and their messenger RNAs, lymphoid tumor cells that express Fc receptors are powerful and unique experimental models for investigating the developmental biology, regulation and function of lymphocyte Fc receptors.     

Coexistence of two lymphomas with distinctive histologic, ultrastructural, and immunologic features.

The unusual coexistence of two distinct lymphomas in 44-year-old woman is described. Nodular, poorly differentiated lymphocytic lymphoma and diffuse histiocytic lymphoma were present in separate sites and were readily distinguished both histologically and ultrastructurally. In addition, the lymphocytic lymphoma was shown to be derived from complement receptor B lymphocytes of follicular center cell type, whereas the histiocytic lymphoma cells were devoid of complement receptors, receptors for IgG (Fc receptors), and surface immunoglobulin. Despite intensive chemotherapy and radiation therapy, the patient died within eight months of the initial diagnosis. Although histiocytic lymphoma was widely disseminated at autopsy, lymphocytic lymphoma was not found. Presumably the histiocytic lymphoma was refractory to therapy, in contrast to the lymphocytic lymphoma, which was selectively eradicated.     

EA rosette-forming lymphoid cells in chickens: specificity of the Fc receptor and its relationship to other surface antigens.

The receptor for erythrocyte-antibody (EA) complexes on the surface of chicken lymphoid cells was investigated using a rosette assay. The chicken EA receptor binds chicken immunoglobulin of the IgG class but not the IgM class. Binding to the EA receptor is dependent upon the Fc region of the immunoglobulin. No receptor for complement analogous to the mammalian C3b receptor was demonstrated on chicken lymphoid cells using the rosette assay. Inhibition studies utilizing immunoglobulins from several species demonstrated that chicken spleen cells do not bind mammalian immunoglobulin but may bind immunoglobulin of other avian species (turkey and duck) and a reptilian species (turtle). The chicken EA receptor is distinct from cell membrane bound immunoglobulin light chains, bursa-specific antigens and thymus-specific antigens. The receptor for EA complexes on chicken lymphoid cells is compared with the Fc receptor on mammalian lymphoid cells in the light of these observations.     

Characterization of Non-Hodgkin''s Lymphomas Using Multiple Cell Markers: Immunologic, Morphologic, and Cytochemical Studies of 72 Cases

Tissues from 72 cases (87 specimens) of various non-Hodgkin's lymphomas were analyzed for cell markers using multiple techniques. Cell suspensions were evaluated for E, EAC, and IgGEA rosette forming cells; Fc receptor cells; and surface immunoglobulin bearing cells. Cryostat section studies topographically defined EAC binding cells. Cytochemical determinations and immunoperoxidase methods for detection of intracellular immunoglobulin and lysozyme complemented other techniques in evaluating infiltrates containing large neoplastic cells. B-cell malignancies comprised 58 cases (80%) of this series and included well and moderately well differentiated lymphocytic lymphomas (10/10); nodular (23/23) and diffuse (10/18) poorly differentiated lymphocytic lymphomas; and lymphomas of mixed lymphocytic-“histiocytic” (3/3), “undifferentiated” (3/3), and “histiocytic” (9/13) types. Nodular lymphomas were characterized as B-cell neoplasms but also revealed a prominent population of T lymphocytes (39 ± 12%). Alkaline phosphatase activity, a cytochemical marker for lymphoid cells of follicular cuffs, was most consistently observed in B-cell lymphomas of moderately well differentiated lymphocytic type (4/6 cases). In some diffuse lymphomas, cryostat section studies (EAC rosettes) suggested a pre-existing nodular proliferation. One unusual B-cell lymphoma of large cell type exhibited IgGEA rosette formation and a strong receptor for the Fc portion of IgG. Ten lymphomas (14%) were of T-cell type and were represented by cases of diffuse poorly differentiated lymphocytic lymphoma (5/18, including 3 lymphoblastic lymphomas), Sézary syndrome (1), mycosis fungoides (1), and a cytologically distinctive large cell (“histiocytic”) lymphoma (3/13). Acid phosphatase activity was a consistent marker for the T-cell malignancies, some of which also revealed α-naphthyl butyrate esterase activity. No true histiocytic lymphomas were detected. Three cases of diffuse poorly differentiated lymphocytic lymphoma and one “histiocytic” lymphoma were null.     

Difference in the Effect of Immobilized Ligands on the Fc and C3 Receptors of Mouse Peritoneal Macrophages in Vitro

The function of the Fc and C3 receptors on the free surface of normal and endotoxin activated mouse peritoneal macrophages seeded on glass bound antibody--antigen (AbAg) complexes or complement was examined. We found that the glass bound AbAg complexes interfered with attachment and internalization of particles recognized by the Fc receptor. The C3 receptor function in these cells was not affected. On the other hand, normal and activated macrophages seeded on glass bound complement showed no change in the function of the C3 and the Fc receptors.     

The effect of complement in adherent immune complexes on Fc and C3 receptor expression in human monocytes.

The effect of complement in surface-bound immune complexes on the expression of Fc and C3 receptors in membranes of adherent human monocytes was examined. Monocytes were isolated from mononuclear leucocyte preparations by adherence to substrates containing fibrin, fibrin with immune complexes (containing rabbit IgG antibodies), or fibrin with immune complexes and mouse complement. Fc or C3 receptors on the top or exposed surface of the monocytes were detected by rosette formation with sheep erythrocytes coated with IgG (EA) or IgM and complement (EAC). Monocytes adherent to surface-bound immune complexes exhibited an absence of EA rosette-forming ability without any change in EAC rosettes. This specific loss of Fc receptor function was induced more easily in freshly-isolated monocytes than in cells maintained in suspension culture for up to 7 days. The presence of complement in the immune complex substrates did not reverse the decrease in Fc receptors seen with freshly-isolated or cultured monocytes. Monocytes adherent to immune complexes and complement exhibited a decrease in C3 receptor function. This decrease was more readily induced in cells cultured for three days in the presence of serum than in freshly-isolated monocytes. Experiments performed with EAC or immune complex substrates relatively enriched in C3b or C3bi indicated that C3b in the substrate induced a decrease in monocyte C3b receptors and C3bi led to a decrease in C3bi receptors. No evidence was found for C3d receptors on the human monocytes although these receptors on a subpopulation of human lymphocytes appeared to be altered in a similar fashion.