A rosette assay for the determination of C 1 q receptor-bearing cells.

A rosette assay for the identification of cells with receptors for C 1 q is described. Glutaraldehyde-treated bovine erythrocytes bound C 1 q specifically, and the reagent thus prepared provided a valid indicator for rosette formation mediated by C 1 q receptors. The presence of these receptors on the membrane of a subset of human peripheral lymphocytes (mainly non-G cells) and on B-derived lymphoblastoid cells was confirmed. Rosette formation was dependent on the number of C 1 q molecules bound per indicator cell and was specifically inhibited by soluble native C 1 q and pepsin-resistant C 1 q fragments. These data, together with the reduced binding activity of C 1 r-C 1 s-associated C 1 q, indicated that the C 1 q binding sites for lymphoid membranes are expressed on the collagen-like moiety, C 1 q rosette formation provided a simple new procedure for fractionation of human lymphocyte populations and separation from phagocytes that do not express receptors for C 1 q.     

T-lymphocyte rosette inhibition titering of antisera by direct microassay

A 60-sample micro-rosette inhibition assay for determining the 100% rosette inhibitory titers of heterologous antisera is described. The assay is performed in histocompatibility trays, under oil, using frozen-thawed thymocytes or peripheral blood lymphocytes as the rosette forming lymphoid cells. Antisera dilutions are incubated with lymphocytes for an appropriate period, either with or without added complement; SRBC are inoculated into each well, and rosettes formed by centrifugation of the trays at 200 × g. Following centrifugation, glutaraldehyde is added to each well to fix rosettes to the well bottom and the plates inverted to allow unbound SRBC to fall away. One hundred percent rosette inhibition is determined by low power microscopic examination of the inverted wells. Highly reproducible in-assay (±4% average standard error) and between-assay (±9%) inhibition values are obtained which correlated well (r = 0.99) with values calculated by more conventional methodology.     

Detection of antigen specific rosette formation with the FACS

Donors previously sensitized to conventional antigens PPD and KLH were evaluated for their antigen binding responses, utilizing a rosette forming technique with antigen-conjugated autologous erythrocytes. Reactivity is directly correlated with prior sensitization. Furthermore, antigen specificity is suggested by inhibition of rosette formation by prior incubation with the relevant antigen. The frequency of RFCs detected cytofluorometrically was compared with conventional fluorescent microscopy determinations. RFCs detected in this manner were identified as antibody armed monocytes by cell depletion and histochemical studies. The usefulness of the rosette forming technique for the routine evaluation of donor immunity is discussed.     

Control of interleukin 1 (IL-1) activity. I. Inhibition of IL-1 activity by soluble immune response suppressor (SIRS) in vitro.

Soluble immune response suppressor (SIRS), a nonspecific inhibitor of cellular and humoral immune responses and cellular proliferation, reversed IL-1-induced inhibition of autologous rosette formation by thymocytes. In addition, SIRS prevented the IL-1-induced increase in resistance of thymocytes to the lytic action of hydrocortisone. Kinetic experiments showed that the action of SIRS on thymocytes was rapid (less than 15 minutes), although a longer time was required to exert protective effects on thymocytes. SIRS also inhibited the stimulation of thymocyte proliferation induced by Con A and IL-1 a costimulatory assay of IL-1 activity. Moreover, SIRS inhibited the IL-1-stimulated expression of complement receptors on neonatal B cells. The inhibitory effects of SIRS were selectively directed towards IL-1, since SIRS did not interfere with induction of LAK cells by IL-2, and did not reverse inhibition of autologous rosette formation induced by factors other than IL-1, such as IL-4, a proline rich polypeptide and lactoferrin. The results presented in this report demonstrate that SIRS may be a selective inhibitor of IL-1 activity with respect to T and B cells, rendering them unresponsive to IL-1 activation and/or maturation signals.     

Modulation of E rosette forming activity of human lymphocytes by rosette inhibiting factor (RIF) ann rosette restoring factor (RRF).

The mechanism of action of RIF and RRF on spontaneous rosette formation by human T lymphocytes with SRBC was studied on cells isolated from healthy donors and from patients with active pulmonary tuberculosis. E rosette formation by lymphocytes from healthy donor is inhibited in vitro by RIF. The effect of RIF is reversible since the primary activity of lymphocytes can be restored by contact with RRF. On the other hand, E rosette formation by lymphocytes from tuberculous patients by means of RRF can be depressed by RIF to the starting level. It was found that prostaglandin synthetase inhibitors (indomethacin and RO 20-5720) prevent inhibition or restoration of E rosette activity by RIF and RRF, respectively.     

Inhibition of complement-dependent lymphocyte rosette formation by sera of patients with chronic glomerulonephritis.

It has been demonstrated that activated C3 products might bind to lymphocyte C3 receptors and inhibit subsequent complement-dependent lymphocyte rosette formation. Sera from patients with various types of chronic glomerulonephritis (GN) have been tested in a complement-dependent rosette inhibition assay using normal donors' lymphocytes as reacting cells. Control subjects consisted of healthy donors and patients with miscellaneous renal and general diseases. Most sera of membranoproliferative GN and of systemic lupus erythematosus, and two-thirds sera of focal glomerolosclerosis patients, significantly inhibited rosette formation. Only 15-40 percent sera of patients with other types of GN were inhibitory. Serum inhibiting activity usually correlated with low serum C3 level (P less than 0.0005), although inhibition could be observed with normal serum C3. However, no correlation was found between a patient's own complement-dependent lymphocyte rosette count and his serum inhibitory activity. These results extend previous findings and suggest that the complement-dependent rosette inhibition assay can be used routinely to detect serum activated complement components either free or bound to immune complexes.     

A new photometric method for the quantitation of Fc receptors for murine IgG1 on human monocytes and cell lines

We have previously shown a polymorphism of human Fc receptors for mouse IgG1 using an EA rosette technique in which human erythrocytes sensitized with a murine IgG1 monoclonal antibody against glycophorin A acted as indicator cells. We now describe a method to quantitate this EA rosetting using the pseudoperoxidase activity present in erythrocytes. This photometric assay allows the sensitive quantitative determination of Fc receptor expression on human monocytes and cell lines. Not only the human Fc receptor for murine IgG1 can be studied in this way, but the method can also be applied to other Fc receptors. An important factor in this type of rosette assay appears to be the amount of negative charge present on the surface of the indicator erythrocytes. Using alcian blue as a probe, we found that this negative charge is higher on human erythrocytes than on sheep erythrocytes, which may contribute to a better signal-to-noise ratio. The method described facilitates the characterization of Fc receptors and permits the rapid screening of monoclonal anti-Fc receptor antibodies.     

A simple and rapid method to distinguish between rosettes and non-specific aggregation of erythrocytes in the rosette assay for human T-lymphocytes.

A method is described for rapid and simple visualization of lymphocytes in the rosette assay for human T-lymphocytes. A drop of acridine orange (pH 7.4) is added to the suspension of rosetted cells and allowed to incubate for 1 min on ice. Nucleic acids are then visualized in a fluorescence microscope by excitation at 430-500 micrometer. Simultaneous semi-illumination with light microscopy allows enumeration of total lymphocytes and the number of lymphocytes rosetted with sheep red blood cells. The central lymphocyte is clearly distinguishable even when it is crowded with red cells, and this makes the differentiation between rosettes and non-specific aggregation of cells easy.     

Detection of IL-2 receptor positive tonsillar lymphocytes by monoclonal antibody and protein A coated erythrocytes

A sensitive rosette method combining staphylococcal protein A coated rabbit red blood cells and the monoclonal antibody anti-Tac was used to search for the presence of interleukin 2 (IL-2) receptor-bearing T lymphocytes in tonsils from patients with chronic tonsillitis. This method revealed the presence of Tac positive T lymphocytes in the tonsils whereas a conventional immunofluorescence technique was unable to do so. To demonstrate that Tac rosette formation resulted in a selective enrichment of IL-2 receptor-bearing T cells, we measured the proliferative response of these cells to exogenous IL-2. The response of Tac rosette positive cells to recombinant IL-2 was always higher than that of the Tac rosette negative or unselected cells, indicating that this rosette method specifically selects T cells expressing IL-2 receptor.     

A method for the quantitative analysis and standardization of interleukin-1 bioactivity

A method is presented for the reproducible quantitation of the biological activity of interleukin 1 (IL-1). This method provides diagnostic tools which give insights into the qualitative aspects of the binding of IL-1 and of the resulting activation of the responder thymocytes; for example, whether the lymphokine and/or the responder population is heterogeneous, or whether a threshold level exists. It establishes under what circumstances the assumptions on which it is based are reasonably adhered to and, consequently, quantitative estimation in the manner it prescribes is justified. It also gives a simple way to calculate both the maximal response attainable for each preparation in an assay and the dilution of a particular preparation that would produce a half-maximal response, the accepted unit of activity of IL-1. This empirical technique provides an improved means of comparing the activities of various preparations of IL-1 in bioassays using various stocks of responder cells and reagents. It should also be applicable to the evaluation of the biological activity of lymphokines in general.     

A simple method for electronic counting of rosette forming human blood lymphocytes.

We investigated the possibility of using a standard electronic laboratory cell counter, of a type which permits manual settings of discriminator and sensitivity levels, for the quantitation of E rosette forming human blood lymphocytes. The electronic counting was performed on rosette specimens prepared from 10 healthy donors, nine lymphoma patients and eight patients with chronic lymphocytic leukaemia (CLL) of B cell type. The simple procedure which was earlier developed for the electronic counting of thymocyte rosettes in guinea-pigs was used and now supplemented with a method for rapid, graphical determination of rosette frequency. The low level of E rosettes in CLL patients was easily distinguished from the higher level seen in the other groups and there was a good agreement between electronic and visual determinations. In samples with a high rosette frequency, the cell counter initially gave 10-30% lower values than when recorded visually. This discrepancy was corrected, however, by a minor adjustment of discriminator level and the introduction of a correction procedure for the presence of double rosettes. We conclude that the electronic counter can well be used for the counting of human rosette forming blood lymphocytes and, due to a high precision and large capacity, this approach would prove to be useful when screening of a large number of samples is required.     

Enhancement of human neutrophil complement receptors: a comparison of the rosette technique with the uptake of radio-labelled anti-CR1 monoclonal antibody.

We have compared the rosette technique (using C3b-coated red cells) with the uptake of a 125I F(ab')2 anti-complement receptor type 1 (CR1) monoclonal antibody (E11) for studying the phenomenon of chemotactic factor (fMLP)-induced complement receptor enhancement (CRE) on human neutrophils. With both methods the dose responses of fMLP were similar with maximal CRE being observed at 10(-7) moles 1(-1). Conversely, the time course of CRE and the effects of disodium cromoglycate (DSCG) on inhibition of enhancement were discrepant. Maximal CRE with the rosette method was observed at 30 min, whereas increased uptake of anti-CR1 had still not reached a plateau after 1 h. DSCG inhibited fMLP-induced CRE, as assessed by the rosette technique, whereas this agent had no effect on increased binding of radio-labelled anti-CR1 antibody. These results suggest that in CRE the increased adherence of C3b-coated red cells to fMLP-stimulated neutrophils, as measured by the rosette technique, is not dependent exclusively on the increased numbers of CR1.     

The occurrence and properties of E rosette inhibitory substance in the sera of malnourished children.

In vitro sheep erythrocyte (E) rosette inhibitory activity was observed in the sera of nine out of 22 (41%) children with kwashiorkor, three of 15 (20%) marasmic children, neither of the two children with marasmic-kwashiorkor and in one of 42 (2%) well nourished control children. Sera of children with kwashiorkor containing the E rosette inhibitory substance did not inhibit in vitro rosette formations by autologous lymphocytes whereas rosette formations by homologous lymphocytes were inhibited. Inhibition of E rosette formation occurred when lymphocytes were pretreated with serum having the inhibitory substance before incubation with sheep red cells, but there was no such inhibition when sheep red cells were pretreated with the same serum before incubation with lymphocytes. The inhibitory substance was observed to be stable at 4 degrees C up to about 1 week and migrated electrophoretically with the alpha-2 globulins. It was digested by papain. It is probable that the E rosette inhibitory substance demonstrated in the present study is attached to markers on T lymphocyte surfaces in some malnourished children thereby making the lymphocytes unreactive in vitro and presumably in vivo as well.     

Partial characterization of the E rosette restoring factor (RRF) from normal human serum

A factor that restores E rosette formation (RRF) was isolated and purified on AMICON membranes. After initial purification, the product showed minimal activity at a concentration of 2 microgram protein per sample. Chromatography on Sephadex G-150 of the partly purified factor caused a further rise in activity to 0.15 microgram per sample. RRF restores normal rosette formation not only by lymphocytes isolated from peripheral blood of patients with active pulmonary tuberculosis, but also stimulated E rosette formation by bone marrow cells. The factor is thermolabile, being completely inactivated at 56 degrees C and partly at 37 degrees C, and is sensitive to freezing and thawing. Its molecular weight is about 10 x 10(3) to 20 x 10(3).     

A microtest for rosette formation.

The microrosette formation test described is carried out in tissue-typing microplates under paraffin oil. Only 8000 lymphocytes in a volume of 2 microliter are required for one assay. This system is very suitable for investigating biopsy-extracted lymphocytes. Furthermore, rosette inhibition can be carried out even when the amount of inhibitory substance tested is minimal.     

Influence of histamine-like drugs and immunomodulators on the spontaneous E rosette formation

The effect of histamine, antihistamine drugs blocking H1 and H2 receptors and immunomodulatory drugs on RF cells was investigated. The test revealed the inhibitory effect of histamine and cimetidine on rosette formation and lack of any effect of antistine. It was found that the "suppressive" activity of histamine may be inhibited by the prior introduction to the culture of histamine H1 and H2-receptor antagonists. Besides, immunomodulatory drugs were observed to inhibit the above mentioned effect of histamine. The involvement of H1 and H2 receptors in the rosette formation was discussed and a suggestion as to the interaction of histamine and immunomodulators was put forward.     

Use of antibody-coated red cells for the sensitive detection of antigen and in rosette tests for cells bearing surface immunoglobulins.

Conditions for an improved chromic chloride method for the attachment of antibody to red cells are described. The method, which is applicable to whole Ig fractions as well as affinity-purified antibodies, is reproducible and has a sensitivity for detection of antigen in the nanogram range. The coated cells may be used in a rosette assay for the detection of cell surface-bound antigens.     

Interleukin-1 decreases the level of thymocytes forming rosettes with autologous erythrocytes: a new method of determination of interleukin-1 activity

It was demonstrated that human, recombinant Il-1 significantly inhibited formation of autologous rosettes by thymocytes. In addition, Il-1 increased the percentage of thymocytes resistant to hydrocortisone. Il-2 was not active in both tests. A new method of determination of Il-1 activity, based on the inhibition of the autologous rosette formation by Il-1 is described. The activity of Il-1 in the supernatant from macrophage cultures, stimulated with carbonyl iron powder, was calculated. The lack of Il-2 interference and no necessity to handle radioactive materials, are the advantages of the new method.     

The relationship between immune rosette forming cells and plaque forming cells in the lizard, Calotes versicolor

The specificity of immune rosette forming cells was assessed using the in vitro blocking of rosette formation with sonicated erythrocyte fragments. An appreciable degree of inhibition of rosette formation against appropriate erythrocytes was observed. The ability of immune rosette forming cells to form plaques was analyzed by separating immune rosettes on a fetal calf serum (FCS) gradient or by separating immune spleen cells on Ficoll-isopyenic gradient. The majority of rosette forming cells were found in a fraction different from that of plaque forming cells. We confirmed these results using a rosette-plaque assay. The data indicate that the majority of antigen-binding cells do not secrete antibody at the peak of immune response.     

Thymic hormonal activity on human peripheral blood lymphocytes, in vitro. I. Reciprocal effect on T and B rosette formation.

One hour incubation with the thymic extract TP-1 induced reciprocal effect on B and T rosette formation in lymphocytes of human peripheral blood. The percentage of mouse erythrocyte rosette-forming cells among lymphocytes of chronic lymphatic leukaemia was decreased by TP-1 from 54.5% to 27.1% (P < 0.001). No such effect was observed in healthy adult or cord blood lymphocytes. On the other hand, the percentage of sheep erythrocyte rosette forming cells increased significantly after TP-1 treatment, but only under conditions of active rosette formation and not in the total rosette assay. This increase was highly significant in three conditions with relative deficiency of cell-mediated immunity: newborns (17.1 to 28.3%), cancer patients (24.5 to 31.7%) and patients with lepromatous leprosy (19.8 to 31.8%). Only a small increase was noticed in healthy adults. A similarly prepared spleen extract was not active in either B or T rosette assays.