Clin. exp. Immunol. (1977) 27, 425-431.
Characterization of human adenoid cells using surface and functional markers for lymphocyte subpopulations BRITTA RYNNEL-DAGOO, ERNA MOLLER & ELIZABETH WATERFIELD Department of Otolaryngology and Transplantation, Immunology Laboratory, Huddinge Hospital, Huddinge, Sweden
(Received 3 September 1976) SUMMARY
Adenoid lymphocytes from children undergoing adenoidectomy were compared with blood cells from the same children using techniques for identifying T cells and B cells. A high proportion of adenoid lymphocytes were immunoglobulin positive cells. Of these only a minor fraction carried receptors for the Fc part of IgG. Adenoid B lymphocytes respond poorly if at all to polyclonal B-cell activators, such as LPS or PPD, which show a different reactivity compared to human splenic cells. The response to anti B2-microglobulin was also different; blood cells responded better than adenoid cells. Thus distinct subpopulations of B lymphocytes reside in different lymphoid organs. The adenoid lymphocyte reactivity might reflect their function in the defence mechanism against infections.
INTRODUCTION Attention has been drawn in recent years to the involvement of the lymphatic tissues in the nasopharyngeal mucosa, the tonsils and the adenoid in the defence mechanism against pathogenic microorganisms. It is now well established that there exist subpopulations of both T and B lymphocytes with varying degrees of differentiation and possibly function. These have been extensively studied in murine systems (Transplantation Reviews 11, 1972) using cell surface as well as functional markers. The same criteria have been used to delineate human subpopulations (Transplantation Reviews 16, 1973). The most reliable surface marker, when attempting to classify a lymphocyte as a B cell, is the presence of immunoglobulin on the cell surface (Moller, 1961). It is also known that certain lymphocytes have a receptor for the Fc portion of IgG which has been extensively used as a marker (Dickler & Kunkel, 1972; Basten et al., 1972; Fr0land & Natvig, 1973). The ability of human lymphocytes to form rosettes with uncoupled sheep red blood cells is well known as a T cell marker (Bianco et al., 1971; Jondal, Holm & Wigzell, 1972, Fr0land & Natvig, 1973). In the mouse and the human, ligands capable of stimulating lymphocytes polyclonally have been used to characterize cell subpopulations. Con A and PHA have been shown to preferentially activate T cells and pokeweed mitogen has been described as containing both T- and B-cell mitogens. LPS from E. coli, dextran sulphate, PPD and anti B2-microglobulin have been shown to be capable of inducing blast transformation and polyclonal antibody synthesis, and thus can be classified as B-cell mitogens (Transplantation Reviews 11 and 16; Coutino, Moller & Richter, 1974; Ringden & Moller, 1975). We wanted to use available surface as well as functional markers to characterize the immunocompetent cells in the adenoid in order to elucidate the physiological role of this tissue. MATERIALS AND METHODS Preparations of adenoid lymphocytes. Adenoids were obtained at adenoidectomy from children between 3 and 8 years of age. The excised tissue was immediately placed in PBS, cut into small pieces and passed gently through a steel screen. Correspondence: Dr B. Rynnel-Dagoo, Department of Otolaryngology and Transplantation, Immunology Laboratory,
Huddinge Hospital, S-141 86, Huddinge, Sweden. D
425
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Clumps were allowed to sediment and the resulting single cell suspension was separated as according to B0yum (1968). Antisera. Rabbit IgG anti-human B2-microglobulin (anti-B2m) (lot number 055 Dakopatt reagents, Copenhagen, Denmark) was dialysed, heat inactivated and used in a final concentration of 1:4. Mitogens. Phytohaemagglutinin (PHA) was obtained from Wellcome Ltd, England. Con A was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Lipopolysaccharide (LPS) from E. coli 0 55:85 were prepared by phenol water extraction (courtesy of Professor T. Holme, Department of Bacteriology, Karolinska Institute, Stockholm). Purified protein derivate (PPD) from tuberculin was obtained from the State Serum Institute, Copenhagen, Denmark. Dextran sulphate (DXS) with 17%4 sulphur-substitution, 50,000 mol. wt (Pharmacia Fine Chemicals, Uppsala, Sweden) and Pokeweed mitogen (PWM) were obtained from Professor G. Moller, Division of Immunobiology, Karolinska Institute, Stockholm, Sweden). The mitogens were diluted in medium for microcultures. Culture conditions. The cells were cultured in medium, prepared according to Mishell & Dutton (1967) with 10% heatinactivated human AB serum added. For measurement of DNA synthesis, quadruplicates of 0 25 x 106 cells in 0 1 ml were cultured in microtitre plates (Falcon 3040). The cultures were incubated at 370C in a humidified 5% CO2 in air atmosphere. Mitogens were diluted in medium and added in 0 1 ml volumes to microculture plates. Measurement of DNA synthesis. 1 pCi of [3H]thymidine in 0 05 ml of PBS was added/well 24 hr prior to harvest. The cells were harvested in a Skatron harvesting machine (Lierbyen, Norway). Radioactivity was measured in an Intertechnique (Nanoteknik, Sweden) scintillation counter. Results were computed and plotted in a Hewlett-Packard computer. All data are presented as arithmetic means+ standard errors of four individual cell cultures. Assay fbr T lymphocytes. 0 25 ml of a 1% suspension of washed SRBC in heat-inactivated foetal calf serum absorbed with SRBC was mixed with 0 25 ml lymphocytes (l x 106) suspended in medium. After 30 min incubation at 37°C the mixture was centrifuged at 100 g for 10 min and kept on ice overnight. Cells binding three or more SRBC were considered positive rosette-forming cells (SRBC-RFC). (At least 200 lymphocytes were screened). Assay for B lymphocytes. For enumeration of B cells, the surface membrane immunoglobulin marker (SmIg) was used. Washed lymphocytes were incubated for 30 min at 20°C in 0-2 ml fluorescein-labelled polyvalent rabbit anti-human Ig serum (Behringwerke, Germany) diluted 1:8 in PBS. After washing three times in cold PBS containing 0-2%/ sodium azide, the cells were kept on ice until examination for membrane fluorescence. At least 200 cells were counted in a Zeiss fluorescence microscope. Assay for lymphocytes with receptors for the Fc portion of IgG. Washed lymphocytes were resuspended in cold PBS at a concentration of 4x 106 cells/ml. To 0 5 ml of this was added 0 1 ml of a 1% suspension of rabbit anti-sheep Ig-coupled sheep red blood cells in cold PBS. The mixture was then centrifuged for 10 min at 100 g in the cold, and allowed to stand on ice for at least 45 min before counting. Cells binding more than four red blood cells were counted as positive Fc-RFC. Assay for lymphocytes with receptors for C'3. Washed lymphocytes were resuspended in cold PBS at a concentration of 4 x 106 cells/ml. To 0 5 ml of this was added 0 1 ml of a 1% suspension of rabbit andti-sheep IgM and A/Sn C'-coupled sheep red blood cells in a cold PBS. The cells were then treated as for Fc-RFC. Cells binding more than four red blood cells were counted as a positive C'3-RFC. Enrichment for B cells. Lymphocytes were rosetted as described above. After 2 hr on ice, the pellet was gently resuspended and layered onto Lymphoprep gradients. After centrifugation, unrosetted cells were removed from the interface and washed three times in PBS before use.
RESULTS Characterization of adenoid lymphocytes using surface markers We have investigated the proportions of T and B cells in adenoids using rosette and immunofluorescence techniques. In adenoids from twenty-three children undergoing adenoidectomy, we found 52-7+ 1-6/o Ig positive cells in the adenoid and 25-9+ 13% in the blood. The proportion of spontaneous rosette-forming cells were 31-7+2-0% in the adenoid and 47±5+ 1.6% in the blood. The proportion of cells binding IgG-coated sheep red blood cells in adenoid lymphocytes from seven children was found to be 13-5+ 1.3 and the mean value for cells binding C'3 for the same children was 50-8+ 330/. In four experiments using B-enriched lymphocytes we found a very low value of Fc-RFC, but an increased number of C'3-RFC (Tables 1 and 2). Characterization of adenoid lymphocytes using functional markers for T and B cells We have investigated the effects of several known mitogens on lymphocytes obtained from both adenoid and blood. Rabbit anti-human B2 microglobulin as well as LPS from E. coli, PPD and DXS have been shown to have mitogenic properties for murine B cells (Andersson, Sj6berg & Moller, 1972; Moller & Persson, 1974; Gronowicz & Coutino, 1975). Further studies have shown that anti-B2-
Characterization of human adenoid cells
427
TABLE 1. Surface markers on adenoid lymphocytes
Patient
Ig positive cells
Fc-RFC
C'3-RFC
SRBC-RFC
1 2 3 4 5 6 7
55*4 54 5
97 12-6 110 20-4 12 7 15-4 13-2 13-5+ 1-3
51-8 60-2 45 5 47-6 58-8 35 6 56 0
38-1 47 5 38-7 30 4 36-9 33-3 19-6
50*8+3 3
34-9+3-2
49-8 50-0 49-0 34-3 52 0 49-3+2-6
TABLE 2. Surface markers in unseparated and B-cell enriched suspensions of adenoid lymphocytes
Ig positive cells 1 U E 2 U E 3 U E 4 U E
Fc-RFC
C'3-RFC
SRBG-RFC
20-7 2-6 14-3 1-5 22-0 40 12-0 05
62-9 35 9 46-3 63-1 35 0 49-0 35*0 39-0
23*5
51-9 84-0 50-2 83-7 46-0 850
57*0 77*0
05 34 0 2-5 42-0
5*0 27-0 1-5
U = Untreated cells; E = B-cell enriched suspension.
90
7
0
E "I
SS-
z
3
4 Time (days)
5
FIG. 1. Kinetic study of DNA synthesis with three different concentrations of adenoid lymphocytes (a) 5x 106/ml; (i) 2 5x 106/ml and (v) 1 5x 106/ml, all induced by anti-B2 microglobulin.
428
Britta Rynnel-Dagi6, Erna Miller & Elizabeth Waterfield TABLE 3. DNA synthesis in lymphocytes from adenoid and in blood lymphocytes stimulated with anti-B2-microglobulin Net counts
ct/min (x 10- 3)
Patient
Adenoid
Blood
1 2 3 4 5 6 7
213+20 219+25 410+59 281+10 80-9+57 599+24 93+ 12
344+09 552+ 17 1261+11 639+13 913+23 458+44 1274+ 16
Mean background was for adenoid cells 14 2+ 3 4 ct/min and fot blood 2 2+ 0 4 ct/min.
microglobulin acts as a polyclonal B-cell activator for human lymphocytes from blood (Ringden & Mdller, 1975), lymph node, spleen and tonsil (Ringden, 1976). In order to determine the optimal conditions for mitogenic stimulation of adenoid lymphocytes by anti-B2-microglobulin, lymphocytes were adjusted to 5, 2-5 and 1.5 x 106 cells/ml in Mishell-Dutton medium+ 10% ABS, set up with various dilutions of anti-B2-microglobulin and harvested on days 3, 4 and 5 respectively. A representative experiment is shown in Fig. 1. As can be seen, the response to anti-B2-microglobulin is highest on day 3 with all cell concentrations. The optimal response is obtained with 2-5 x 106 lymphocytes per microculture well. In our laboratory optimal responses of blood lymphocytes to anti-B2-microglobulin were obtained with 1.5 x 106 cells per ml. Hence for experiments with blood lymphocytes, this concentration was used. The response to anti-B2-microglobulin of adenoid lymphocytes and blood lymphocytes from seven patients was compared. The results are shown in Table 3. The response is significantly higher in cells from blood than in cells from adenoids, in spite of the fact that the proportion of B cells is higher in adenoids than in blood. Recent experiments have indicated that human spleen cells are highly responsive to LPS, whereas blood lymphocytes are unresponsive (Ringden & Mbller, 1975). This was confirmed in our experiments. No increase in DNA synthesis was noted when blood lymphocytes were cultivated with LPS in doses ranging from 1 ug per ml to 100 ug per ml. In five out of nine cases, adenoid lymphocytes were refractory to stimulation with LPS. However, in the remaining four cases net increases in 3H incorporation between 5000 and 12,000 ct/min were noted. We have no explanation for this discrepancy and these patients are subject to further investigation. In these four cases the optimal concentration of LPS was 100 pg per ml, consistent with conditions where LPS is highly mitogenic. Dextran sulphate acts as a PBA in the mouse system, mainly affecting immature cells which proliferate extensively on exposure to the mitogen, but are not activated to high rate antibody production (Gronowicz & Coutino, 1975). It has not yet been shown to be a PBA for human cells. No response was obtained with either adenoid lymphocytes or lymphocytes from blood using concentrations of Dextran sulphate ranging from 10 yg/ml to 1 mg/ml. In two experiments we used adenoid lymphocytes without preparation of Lymphoprep and still had no response. Like LPS, PPD has been shown to induce DNA synthesis in human spleen cells. However, no response has yet been observed in blood lymphocytes on the third day of culture (Ringden, 1976). We tested doses ranging from 10-200 pg/ml and found a slight stimulation with 100 jg/ml on day 3. Cells from two patients gave net count increase around 16,000. WVith lymphocytes from adenoid the mean value was 6000. Cells from three patients did not respond at all, whereas cells from five patients gave net counts increases of 8-19,000. There was no correlation between the response in blood and in adenoid in the same patient.
Characterization of human adenoid cells ,.e
42
...
429
..
-
36g30-~24
PHA ANTI-B2 LPS. PPD Con A PWN FIG. 2. DNA synthesis in human adenoid cells (open columns) and blood lymphocytes (stippled column) induced by different mitogens. *
Stimulation to DNA synthesis by PHA, Con A and PWM gave similar values for blood and for adenoid. PHA was tested in doses ranging from 0 001-1 fig, Con A from 0 02-1 ,ug and PMW from 0-01250,g/ml. Optimal responses were invariably observed at the same mitogen concentration for both blood and adenoid cells (Fig. 2). DISCUSSION It is now quite clear that different subpopulations of both T and B lymphocytes reside in different organs. Lymphocytes in the adenoid being constantly exposed to natural antigens in nasopharynx have been shown to contain a high proportion of Ig positive cells, slightly lower than that found in the human spleen (Ringden & Moller, 1975). This agrees with the proportion of villous surface tonsil lymphocytes detected by scanning electron microscopy (Tabata et al., 1975). The proportion of smooth surface lymphocytes observed by the same method is also in accordance with our results. The presence of Fc and C'3 receptors has, in murine systems, been shown to characterize bone marrowderived cells and monocyte-macrophage cells (Transplantation Review, 11). In man, most Ig positive lymphocytes in blood also have Fc receptors. In human foetal spleen, however, 80% of the lymphocytes have surface Ig of various classes but only 20% of those had detectable Fc receptors (Jondal, Wigzell & Aiuti, 1973). With the present method for demonstration of Fc binding adenoid lymphocytes, we found low values of cells carrying Fc receptor compared to the high value of Ig-positive cells. This is in accordance with the findings of Fr0land & Natvig (1973) who found low values in both tonsil and adenoid. Recently Samarut, Brochier & Reuillard (1976) found a nearly total absence of Fc-RFC in highly purified lymphocytes from tonsils. This indicates that many B cells bearing surface Ig lack Fc receptor detectable by rosette formation. In four experiments we found 4% Fc-binding cells in suspensions enriched for B cells. Samarut et al. (1976) have clearly shown that Fc receptors in humans are present on different lymphocyte populations and therefore can not be regarded as a specific marker of any one of these populations. Such polyclonal B-cell activators as LPS from E. coli, dextran sulphate, PPD and anti-B2-microglobulin stimulate subpopulations of murine B cells which differ in their degree of differentiation (Gronovicz & Coutino, 1975). Cells responsive to dextran sulphate are thought to be more primitive, LPS-responsive cells to be more mature, and PPD-responsive cells to be end cells, incapable of being restimulated by either LPS or PPD. Since a high proportion of cells in the adenoid are capable of activity synthesizing Ig (Smith, Sherman & Newcomb, 1974), it would be expected that the proportion of immature cells able to respond to DXS could be correspondingly low. However, cord blood lymphocytes are also refractory to stimulation by DXS (unpublished observations). It has been suggested that macrophages are essential for activation
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Britta Rynnel-Dag56, Erna Miller & Elizabeth Waterfield
by DXS (Ulla Persson, personal communication) thus we carried out two experiments with unseparated adenoid cells, but no increase in DNA synthesis was noted. LPS, PPD and anti-B2-microglobulin have been shown to activate human spleen cells to DNA synthesis and antibody production (Ringden, 1976). Similar proportions of Ig-positive cells are found in human spleen and in the adenoid. However, the mitogenic responses differ considerably. Adenoid cells, like blood lymphocytes, respond poorly if at all to either LPS or PPD on day 3. Spleen cells, on the other hand, are activated to DNA synthesis by both mitogens. Anti-B2-microglobulin stimulates splenic lymphocytes to DNA synthesis to a higher extent than blood lymphocytes. Adenoid lymphocytes mount a response which, in general, is lower than that of blood lymphocytes. This raises the question as to whether different subsets of B cells are present in the adenoid and spleen. It could however be possible that the B cells of the adenoid had been activated to such an extent prior to extraction due to their contact with natural antigens in the nasopharynx, that they are incapable of further activation by other mitogens. However, the anti-B2-microglobulin results would appear to disagree with this. We have also found that adenoid lymphocytes respond well to both PHA, Con A and PWM, and there is no significant difference between the responses in adenoid and blood. It has been suggested that a T-cell functional deficiency exists in the nasopharyngeal lymphoid organs (Hoffman, Schmidt & Oettgen, 1973, Watanabe et al., 1974) but our results do not give support to this notion. Adenoid lymphocytes have been shown to produce IgA and IgG spontaneously as well as after stimulation by PWM (Smith, Sherman & Newcourt, 1974). Tonsil lymphocytes have been shown to be stimulated in vitro with diphtheria toxoid and polio virus and it has been possible to measure production of Ig as increasing levels of neutralizing antibody (Platts-Mills & Ishizaka, 1975; Sloyer, Veltri & Sprinkle, 1973). Adenoid lymphocytes from children older than 4 years of age have been shown to produce larger amounts of IgG than cells from younger children. (Ishikawa, Wicher & Arbesman, 1972) which possibly would indicate that with time the lymphocytes develop a higher degree of differentiation and capability to respond to antigenic stimulation. Furthermore, if the cells are activated by natural bacterial antigens, such as haemophilus influenzae, which has been shown to contain mitogenic moieties, an increase in DNA synthesis is noted (unpublished observations). From what has been stated above functional characteristics of lymphocytes in the adenoid might very well reflect their immunological history. The adenoid is thus an immunocompetent organ containing cells supposed to be directly stimulated by natural antigen. A high proportion of lymphocytes are Ig-positive cells, which by several indirect methods have been shown to produce large amounts of IgG, IgM and especially IgA (Ishikawa et al., 1972). XWe have been able to show intracellular immunoglobulin in a fraction of blast cells stimulated with PWM (unpublished observation). The immunocompetent tissue in the nasopharynx is active during childhood, during a period when many children have recurrent attacks of infections of the upper respiratory tract. The adenoid has for a long time been regarded as a focus of such infections rather than part of the first line of defence. Our results have shown that, even during a time when the children are suffering from recurrent infections, the cells of the adenoid are still capable of being activated, and are thus conceivably a valuable component of the immune system. REFERENCES ANDERSSON, J., SJOBERG, 0. & MOLLER, G. (1972) Mitogens as probes for immunocyte activation and cellular cooperation. Transplant. Rev. 11, 131. BASTEN, A., MILLER, J.F.A.P., SPRENT, J. & PYE, J. (1972) A receptor for antibody on B lymphocytes. I. A method of detection and functional significance. I. exp. Med. 135, 610. BIANCO, C., NUSSENZWEIG, V., LAY, W.H. & MENDES, N.F. (1971) Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond.), 230, 531.
BoYUM, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. 3. cin. Lab. Invest. 21, Supplement 97. COUTINHO, A., MOLLER, G. & RICHTER, W. (1974) Molecular bases of B cell activation. I. Mitogenicity of native and substituted dextrans. Scand. J. Immunol. 3, 321. DICKLER, H.B. & KUNKEL, H.G. (1972) Interaction of aggregated globulin with B lymphocytes. 3. exp. Med. 136, 191. FROLAND, S.S. & NATVIG, J.B. (1973) Identification of three
Characterization of human adenoid cells different human lymphocyte populations by surface markers. Transplant. Rev. 16, 114. FR0LAND, S.S., NATVIG, J.B. & MICHAELSEN, T.E. (1974) Binding of aggregated IgG by human B lymphocytes independent of Fc receptors. _. Immunol. 3, 375. GREAVES, M. & JANossy, G. (1972) Elicition of selective T and B lymphocyte responses by cell surface binding ligands. Transplant. Rev. 11, 87. GRONOWICZ, E. & COUTINHO, A. (1975) Functional analysis of B cell heterogeneity. Transplant. Rev. 24, 3. HOFFMAN, M.K., SCHMIDT, D. & OETTGEN, H.F. (1973) Production of antibody to sheep red blood cells by human tonsil cells in vitro. Nature (Lond.), 243, 408. ISHIKAWA, T., WICHER, K. & ARBESMAN, E. (1972) Distribution of y (gamma) immunoglobulins in palatine and pharyngeal tonsils. Int. Arch. Allergy, 43, 801. JoNDAL, M., HOLM, G. & WIGZELL, H. (1972) Surfacemarkers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. ]. exp. Med. 136, 207. JONDAL, M., WIGZELL, H. & AIUTI, F. (1973) Human lymphocyte subpopulations: Classification according to surface markers and/or functional characteristics. Transplant. Rev. 16, 163. MISHELL, R.J. & DUTTON, R.W. (1967) Immunization of dissociated spleen cell cultures from normal mice. J. exp. Med. 126, 423. MOLLER, G. (1961) Demonstration of mouse isoantigen at the cellular level by fluorescence antibody technique. J. exp. Med. 114, 415. MOLLER, G. & PERSSON, U. (1974) Mitogenic properties of rabbit anti-human B2-microglobulin for murine B cells. Scand..3. Immunol. 3, 445. PLATTS-MILLS, T.A.E. & ISHIZAKA, K. (1975) IgG and IgA
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diphtheria antitoxin responses from human tonsil lymphocytes. J. Immunol. 114, 1058. RINGD&N, 0. & MOLLER, E. (1975) B cell mitogenic effects of rabbit anti-human B2 microglogulin for human lymphocytes. Scand. J. Immunol. 4, 171. RINGDEN, 0. (1976) Activation of human lymphocyte subpopulations by rabbit anti-human B2 microblobulin and by LPS. Scand. 5. Immunol. 5, (In press.) SAMARUT, C., BROCHIER, J. & REVILLARD, J.P. (1976) Distribution of cells binding erythrocyte-antibody (EA) complexes in human lymphoid populations. Scand. J. Immunol. 5, 221. SLOYER, J.L., VELTRI, R.W. & SPRINKLE, P.M. (1973) In vitro IgM antibody synthesis by human tonsil-derived lymphocytes. 5. Immunol. 111, 183. SMITH, R.S., SHERMAN, N.A. & NEWCOMB, R.W. (1974) Synthesis and secretion of immunoglobulin, including IgA, by human tonsil and adenoid tissue cultured in vitro. Int. Arch. Allergy, 46, 785. TABATA, T., KATSURAHARA, T., ENOMOTO, T., KITASHOJI, N. & TANAKA, S. (1975) Immunological function of human tonsil. Surface topology of human tonsil lymphocytes using the scanning electron microscope (SEM). Acta Otolaryngol. 80, 474. THORSBY, E. & BRATELI, A. (1972) A rapid method for preparation of pure lymphocyte suspensions. Histocompatibility Testing (ed. by J. Dausset and Colombani), p. 531. Munksgaard, A/S, Copenhagen. Transplant Rev. (1972) (ed. by G. Moller), Lymphocyte activation by mitogens. 11. Transplant. Rev. (1973) (ed. by G. Moller), T and B lymphocytes in humans. 16. WATANABE, T., YOSHIZAKI, K., YAGURA, T. & YAMAMURA, Y. (1974) In vitro antibody formation by human tonsil lymphocytes. J. exp. Med. 113, 608.
Characterization of human adenoid cells using surface and functional markers for lymphocyte subpopulations BRITTA RYNNEL-DAGOO, ERNA MOLLER & ELIZABETH WATERFIELD Department of Otolaryngology and Transplantation, Immunology Laboratory, Huddinge Hospital, Huddinge, Sweden
(Received 3 September 1976) SUMMARY
Adenoid lymphocytes from children undergoing adenoidectomy were compared with blood cells from the same children using techniques for identifying T cells and B cells. A high proportion of adenoid lymphocytes were immunoglobulin positive cells. Of these only a minor fraction carried receptors for the Fc part of IgG. Adenoid B lymphocytes respond poorly if at all to polyclonal B-cell activators, such as LPS or PPD, which show a different reactivity compared to human splenic cells. The response to anti B2-microglobulin was also different; blood cells responded better than adenoid cells. Thus distinct subpopulations of B lymphocytes reside in different lymphoid organs. The adenoid lymphocyte reactivity might reflect their function in the defence mechanism against infections.
INTRODUCTION Attention has been drawn in recent years to the involvement of the lymphatic tissues in the nasopharyngeal mucosa, the tonsils and the adenoid in the defence mechanism against pathogenic microorganisms. It is now well established that there exist subpopulations of both T and B lymphocytes with varying degrees of differentiation and possibly function. These have been extensively studied in murine systems (Transplantation Reviews 11, 1972) using cell surface as well as functional markers. The same criteria have been used to delineate human subpopulations (Transplantation Reviews 16, 1973). The most reliable surface marker, when attempting to classify a lymphocyte as a B cell, is the presence of immunoglobulin on the cell surface (Moller, 1961). It is also known that certain lymphocytes have a receptor for the Fc portion of IgG which has been extensively used as a marker (Dickler & Kunkel, 1972; Basten et al., 1972; Fr0land & Natvig, 1973). The ability of human lymphocytes to form rosettes with uncoupled sheep red blood cells is well known as a T cell marker (Bianco et al., 1971; Jondal, Holm & Wigzell, 1972, Fr0land & Natvig, 1973). In the mouse and the human, ligands capable of stimulating lymphocytes polyclonally have been used to characterize cell subpopulations. Con A and PHA have been shown to preferentially activate T cells and pokeweed mitogen has been described as containing both T- and B-cell mitogens. LPS from E. coli, dextran sulphate, PPD and anti B2-microglobulin have been shown to be capable of inducing blast transformation and polyclonal antibody synthesis, and thus can be classified as B-cell mitogens (Transplantation Reviews 11 and 16; Coutino, Moller & Richter, 1974; Ringden & Moller, 1975). We wanted to use available surface as well as functional markers to characterize the immunocompetent cells in the adenoid in order to elucidate the physiological role of this tissue. MATERIALS AND METHODS Preparations of adenoid lymphocytes. Adenoids were obtained at adenoidectomy from children between 3 and 8 years of age. The excised tissue was immediately placed in PBS, cut into small pieces and passed gently through a steel screen. Correspondence: Dr B. Rynnel-Dagoo, Department of Otolaryngology and Transplantation, Immunology Laboratory,
Huddinge Hospital, S-141 86, Huddinge, Sweden. D
425
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Britta Rynnel-Dagio, Erna Moller 5 Elizabeth Waterfield
Clumps were allowed to sediment and the resulting single cell suspension was separated as according to B0yum (1968). Antisera. Rabbit IgG anti-human B2-microglobulin (anti-B2m) (lot number 055 Dakopatt reagents, Copenhagen, Denmark) was dialysed, heat inactivated and used in a final concentration of 1:4. Mitogens. Phytohaemagglutinin (PHA) was obtained from Wellcome Ltd, England. Con A was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Lipopolysaccharide (LPS) from E. coli 0 55:85 were prepared by phenol water extraction (courtesy of Professor T. Holme, Department of Bacteriology, Karolinska Institute, Stockholm). Purified protein derivate (PPD) from tuberculin was obtained from the State Serum Institute, Copenhagen, Denmark. Dextran sulphate (DXS) with 17%4 sulphur-substitution, 50,000 mol. wt (Pharmacia Fine Chemicals, Uppsala, Sweden) and Pokeweed mitogen (PWM) were obtained from Professor G. Moller, Division of Immunobiology, Karolinska Institute, Stockholm, Sweden). The mitogens were diluted in medium for microcultures. Culture conditions. The cells were cultured in medium, prepared according to Mishell & Dutton (1967) with 10% heatinactivated human AB serum added. For measurement of DNA synthesis, quadruplicates of 0 25 x 106 cells in 0 1 ml were cultured in microtitre plates (Falcon 3040). The cultures were incubated at 370C in a humidified 5% CO2 in air atmosphere. Mitogens were diluted in medium and added in 0 1 ml volumes to microculture plates. Measurement of DNA synthesis. 1 pCi of [3H]thymidine in 0 05 ml of PBS was added/well 24 hr prior to harvest. The cells were harvested in a Skatron harvesting machine (Lierbyen, Norway). Radioactivity was measured in an Intertechnique (Nanoteknik, Sweden) scintillation counter. Results were computed and plotted in a Hewlett-Packard computer. All data are presented as arithmetic means+ standard errors of four individual cell cultures. Assay fbr T lymphocytes. 0 25 ml of a 1% suspension of washed SRBC in heat-inactivated foetal calf serum absorbed with SRBC was mixed with 0 25 ml lymphocytes (l x 106) suspended in medium. After 30 min incubation at 37°C the mixture was centrifuged at 100 g for 10 min and kept on ice overnight. Cells binding three or more SRBC were considered positive rosette-forming cells (SRBC-RFC). (At least 200 lymphocytes were screened). Assay for B lymphocytes. For enumeration of B cells, the surface membrane immunoglobulin marker (SmIg) was used. Washed lymphocytes were incubated for 30 min at 20°C in 0-2 ml fluorescein-labelled polyvalent rabbit anti-human Ig serum (Behringwerke, Germany) diluted 1:8 in PBS. After washing three times in cold PBS containing 0-2%/ sodium azide, the cells were kept on ice until examination for membrane fluorescence. At least 200 cells were counted in a Zeiss fluorescence microscope. Assay for lymphocytes with receptors for the Fc portion of IgG. Washed lymphocytes were resuspended in cold PBS at a concentration of 4x 106 cells/ml. To 0 5 ml of this was added 0 1 ml of a 1% suspension of rabbit anti-sheep Ig-coupled sheep red blood cells in cold PBS. The mixture was then centrifuged for 10 min at 100 g in the cold, and allowed to stand on ice for at least 45 min before counting. Cells binding more than four red blood cells were counted as positive Fc-RFC. Assay for lymphocytes with receptors for C'3. Washed lymphocytes were resuspended in cold PBS at a concentration of 4 x 106 cells/ml. To 0 5 ml of this was added 0 1 ml of a 1% suspension of rabbit andti-sheep IgM and A/Sn C'-coupled sheep red blood cells in a cold PBS. The cells were then treated as for Fc-RFC. Cells binding more than four red blood cells were counted as a positive C'3-RFC. Enrichment for B cells. Lymphocytes were rosetted as described above. After 2 hr on ice, the pellet was gently resuspended and layered onto Lymphoprep gradients. After centrifugation, unrosetted cells were removed from the interface and washed three times in PBS before use.
RESULTS Characterization of adenoid lymphocytes using surface markers We have investigated the proportions of T and B cells in adenoids using rosette and immunofluorescence techniques. In adenoids from twenty-three children undergoing adenoidectomy, we found 52-7+ 1-6/o Ig positive cells in the adenoid and 25-9+ 13% in the blood. The proportion of spontaneous rosette-forming cells were 31-7+2-0% in the adenoid and 47±5+ 1.6% in the blood. The proportion of cells binding IgG-coated sheep red blood cells in adenoid lymphocytes from seven children was found to be 13-5+ 1.3 and the mean value for cells binding C'3 for the same children was 50-8+ 330/. In four experiments using B-enriched lymphocytes we found a very low value of Fc-RFC, but an increased number of C'3-RFC (Tables 1 and 2). Characterization of adenoid lymphocytes using functional markers for T and B cells We have investigated the effects of several known mitogens on lymphocytes obtained from both adenoid and blood. Rabbit anti-human B2 microglobulin as well as LPS from E. coli, PPD and DXS have been shown to have mitogenic properties for murine B cells (Andersson, Sj6berg & Moller, 1972; Moller & Persson, 1974; Gronowicz & Coutino, 1975). Further studies have shown that anti-B2-
Characterization of human adenoid cells
427
TABLE 1. Surface markers on adenoid lymphocytes
Patient
Ig positive cells
Fc-RFC
C'3-RFC
SRBC-RFC
1 2 3 4 5 6 7
55*4 54 5
97 12-6 110 20-4 12 7 15-4 13-2 13-5+ 1-3
51-8 60-2 45 5 47-6 58-8 35 6 56 0
38-1 47 5 38-7 30 4 36-9 33-3 19-6
50*8+3 3
34-9+3-2
49-8 50-0 49-0 34-3 52 0 49-3+2-6
TABLE 2. Surface markers in unseparated and B-cell enriched suspensions of adenoid lymphocytes
Ig positive cells 1 U E 2 U E 3 U E 4 U E
Fc-RFC
C'3-RFC
SRBG-RFC
20-7 2-6 14-3 1-5 22-0 40 12-0 05
62-9 35 9 46-3 63-1 35 0 49-0 35*0 39-0
23*5
51-9 84-0 50-2 83-7 46-0 850
57*0 77*0
05 34 0 2-5 42-0
5*0 27-0 1-5
U = Untreated cells; E = B-cell enriched suspension.
90
7
0
E "I
SS-
z
3
4 Time (days)
5
FIG. 1. Kinetic study of DNA synthesis with three different concentrations of adenoid lymphocytes (a) 5x 106/ml; (i) 2 5x 106/ml and (v) 1 5x 106/ml, all induced by anti-B2 microglobulin.
428
Britta Rynnel-Dagi6, Erna Miller & Elizabeth Waterfield TABLE 3. DNA synthesis in lymphocytes from adenoid and in blood lymphocytes stimulated with anti-B2-microglobulin Net counts
ct/min (x 10- 3)
Patient
Adenoid
Blood
1 2 3 4 5 6 7
213+20 219+25 410+59 281+10 80-9+57 599+24 93+ 12
344+09 552+ 17 1261+11 639+13 913+23 458+44 1274+ 16
Mean background was for adenoid cells 14 2+ 3 4 ct/min and fot blood 2 2+ 0 4 ct/min.
microglobulin acts as a polyclonal B-cell activator for human lymphocytes from blood (Ringden & Mdller, 1975), lymph node, spleen and tonsil (Ringden, 1976). In order to determine the optimal conditions for mitogenic stimulation of adenoid lymphocytes by anti-B2-microglobulin, lymphocytes were adjusted to 5, 2-5 and 1.5 x 106 cells/ml in Mishell-Dutton medium+ 10% ABS, set up with various dilutions of anti-B2-microglobulin and harvested on days 3, 4 and 5 respectively. A representative experiment is shown in Fig. 1. As can be seen, the response to anti-B2-microglobulin is highest on day 3 with all cell concentrations. The optimal response is obtained with 2-5 x 106 lymphocytes per microculture well. In our laboratory optimal responses of blood lymphocytes to anti-B2-microglobulin were obtained with 1.5 x 106 cells per ml. Hence for experiments with blood lymphocytes, this concentration was used. The response to anti-B2-microglobulin of adenoid lymphocytes and blood lymphocytes from seven patients was compared. The results are shown in Table 3. The response is significantly higher in cells from blood than in cells from adenoids, in spite of the fact that the proportion of B cells is higher in adenoids than in blood. Recent experiments have indicated that human spleen cells are highly responsive to LPS, whereas blood lymphocytes are unresponsive (Ringden & Mbller, 1975). This was confirmed in our experiments. No increase in DNA synthesis was noted when blood lymphocytes were cultivated with LPS in doses ranging from 1 ug per ml to 100 ug per ml. In five out of nine cases, adenoid lymphocytes were refractory to stimulation with LPS. However, in the remaining four cases net increases in 3H incorporation between 5000 and 12,000 ct/min were noted. We have no explanation for this discrepancy and these patients are subject to further investigation. In these four cases the optimal concentration of LPS was 100 pg per ml, consistent with conditions where LPS is highly mitogenic. Dextran sulphate acts as a PBA in the mouse system, mainly affecting immature cells which proliferate extensively on exposure to the mitogen, but are not activated to high rate antibody production (Gronowicz & Coutino, 1975). It has not yet been shown to be a PBA for human cells. No response was obtained with either adenoid lymphocytes or lymphocytes from blood using concentrations of Dextran sulphate ranging from 10 yg/ml to 1 mg/ml. In two experiments we used adenoid lymphocytes without preparation of Lymphoprep and still had no response. Like LPS, PPD has been shown to induce DNA synthesis in human spleen cells. However, no response has yet been observed in blood lymphocytes on the third day of culture (Ringden, 1976). We tested doses ranging from 10-200 pg/ml and found a slight stimulation with 100 jg/ml on day 3. Cells from two patients gave net count increase around 16,000. WVith lymphocytes from adenoid the mean value was 6000. Cells from three patients did not respond at all, whereas cells from five patients gave net counts increases of 8-19,000. There was no correlation between the response in blood and in adenoid in the same patient.
Characterization of human adenoid cells ,.e
42
...
429
..
-
36g30-~24
PHA ANTI-B2 LPS. PPD Con A PWN FIG. 2. DNA synthesis in human adenoid cells (open columns) and blood lymphocytes (stippled column) induced by different mitogens. *
Stimulation to DNA synthesis by PHA, Con A and PWM gave similar values for blood and for adenoid. PHA was tested in doses ranging from 0 001-1 fig, Con A from 0 02-1 ,ug and PMW from 0-01250,g/ml. Optimal responses were invariably observed at the same mitogen concentration for both blood and adenoid cells (Fig. 2). DISCUSSION It is now quite clear that different subpopulations of both T and B lymphocytes reside in different organs. Lymphocytes in the adenoid being constantly exposed to natural antigens in nasopharynx have been shown to contain a high proportion of Ig positive cells, slightly lower than that found in the human spleen (Ringden & Moller, 1975). This agrees with the proportion of villous surface tonsil lymphocytes detected by scanning electron microscopy (Tabata et al., 1975). The proportion of smooth surface lymphocytes observed by the same method is also in accordance with our results. The presence of Fc and C'3 receptors has, in murine systems, been shown to characterize bone marrowderived cells and monocyte-macrophage cells (Transplantation Review, 11). In man, most Ig positive lymphocytes in blood also have Fc receptors. In human foetal spleen, however, 80% of the lymphocytes have surface Ig of various classes but only 20% of those had detectable Fc receptors (Jondal, Wigzell & Aiuti, 1973). With the present method for demonstration of Fc binding adenoid lymphocytes, we found low values of cells carrying Fc receptor compared to the high value of Ig-positive cells. This is in accordance with the findings of Fr0land & Natvig (1973) who found low values in both tonsil and adenoid. Recently Samarut, Brochier & Reuillard (1976) found a nearly total absence of Fc-RFC in highly purified lymphocytes from tonsils. This indicates that many B cells bearing surface Ig lack Fc receptor detectable by rosette formation. In four experiments we found 4% Fc-binding cells in suspensions enriched for B cells. Samarut et al. (1976) have clearly shown that Fc receptors in humans are present on different lymphocyte populations and therefore can not be regarded as a specific marker of any one of these populations. Such polyclonal B-cell activators as LPS from E. coli, dextran sulphate, PPD and anti-B2-microglobulin stimulate subpopulations of murine B cells which differ in their degree of differentiation (Gronovicz & Coutino, 1975). Cells responsive to dextran sulphate are thought to be more primitive, LPS-responsive cells to be more mature, and PPD-responsive cells to be end cells, incapable of being restimulated by either LPS or PPD. Since a high proportion of cells in the adenoid are capable of activity synthesizing Ig (Smith, Sherman & Newcomb, 1974), it would be expected that the proportion of immature cells able to respond to DXS could be correspondingly low. However, cord blood lymphocytes are also refractory to stimulation by DXS (unpublished observations). It has been suggested that macrophages are essential for activation
430
Britta Rynnel-Dag56, Erna Miller & Elizabeth Waterfield
by DXS (Ulla Persson, personal communication) thus we carried out two experiments with unseparated adenoid cells, but no increase in DNA synthesis was noted. LPS, PPD and anti-B2-microglobulin have been shown to activate human spleen cells to DNA synthesis and antibody production (Ringden, 1976). Similar proportions of Ig-positive cells are found in human spleen and in the adenoid. However, the mitogenic responses differ considerably. Adenoid cells, like blood lymphocytes, respond poorly if at all to either LPS or PPD on day 3. Spleen cells, on the other hand, are activated to DNA synthesis by both mitogens. Anti-B2-microglobulin stimulates splenic lymphocytes to DNA synthesis to a higher extent than blood lymphocytes. Adenoid lymphocytes mount a response which, in general, is lower than that of blood lymphocytes. This raises the question as to whether different subsets of B cells are present in the adenoid and spleen. It could however be possible that the B cells of the adenoid had been activated to such an extent prior to extraction due to their contact with natural antigens in the nasopharynx, that they are incapable of further activation by other mitogens. However, the anti-B2-microglobulin results would appear to disagree with this. We have also found that adenoid lymphocytes respond well to both PHA, Con A and PWM, and there is no significant difference between the responses in adenoid and blood. It has been suggested that a T-cell functional deficiency exists in the nasopharyngeal lymphoid organs (Hoffman, Schmidt & Oettgen, 1973, Watanabe et al., 1974) but our results do not give support to this notion. Adenoid lymphocytes have been shown to produce IgA and IgG spontaneously as well as after stimulation by PWM (Smith, Sherman & Newcourt, 1974). Tonsil lymphocytes have been shown to be stimulated in vitro with diphtheria toxoid and polio virus and it has been possible to measure production of Ig as increasing levels of neutralizing antibody (Platts-Mills & Ishizaka, 1975; Sloyer, Veltri & Sprinkle, 1973). Adenoid lymphocytes from children older than 4 years of age have been shown to produce larger amounts of IgG than cells from younger children. (Ishikawa, Wicher & Arbesman, 1972) which possibly would indicate that with time the lymphocytes develop a higher degree of differentiation and capability to respond to antigenic stimulation. Furthermore, if the cells are activated by natural bacterial antigens, such as haemophilus influenzae, which has been shown to contain mitogenic moieties, an increase in DNA synthesis is noted (unpublished observations). From what has been stated above functional characteristics of lymphocytes in the adenoid might very well reflect their immunological history. The adenoid is thus an immunocompetent organ containing cells supposed to be directly stimulated by natural antigen. A high proportion of lymphocytes are Ig-positive cells, which by several indirect methods have been shown to produce large amounts of IgG, IgM and especially IgA (Ishikawa et al., 1972). XWe have been able to show intracellular immunoglobulin in a fraction of blast cells stimulated with PWM (unpublished observation). The immunocompetent tissue in the nasopharynx is active during childhood, during a period when many children have recurrent attacks of infections of the upper respiratory tract. The adenoid has for a long time been regarded as a focus of such infections rather than part of the first line of defence. Our results have shown that, even during a time when the children are suffering from recurrent infections, the cells of the adenoid are still capable of being activated, and are thus conceivably a valuable component of the immune system. REFERENCES ANDERSSON, J., SJOBERG, 0. & MOLLER, G. (1972) Mitogens as probes for immunocyte activation and cellular cooperation. Transplant. Rev. 11, 131. BASTEN, A., MILLER, J.F.A.P., SPRENT, J. & PYE, J. (1972) A receptor for antibody on B lymphocytes. I. A method of detection and functional significance. I. exp. Med. 135, 610. BIANCO, C., NUSSENZWEIG, V., LAY, W.H. & MENDES, N.F. (1971) Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond.), 230, 531.
BoYUM, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. 3. cin. Lab. Invest. 21, Supplement 97. COUTINHO, A., MOLLER, G. & RICHTER, W. (1974) Molecular bases of B cell activation. I. Mitogenicity of native and substituted dextrans. Scand. J. Immunol. 3, 321. DICKLER, H.B. & KUNKEL, H.G. (1972) Interaction of aggregated globulin with B lymphocytes. 3. exp. Med. 136, 191. FROLAND, S.S. & NATVIG, J.B. (1973) Identification of three
Characterization of human adenoid cells different human lymphocyte populations by surface markers. Transplant. Rev. 16, 114. FR0LAND, S.S., NATVIG, J.B. & MICHAELSEN, T.E. (1974) Binding of aggregated IgG by human B lymphocytes independent of Fc receptors. _. Immunol. 3, 375. GREAVES, M. & JANossy, G. (1972) Elicition of selective T and B lymphocyte responses by cell surface binding ligands. Transplant. Rev. 11, 87. GRONOWICZ, E. & COUTINHO, A. (1975) Functional analysis of B cell heterogeneity. Transplant. Rev. 24, 3. HOFFMAN, M.K., SCHMIDT, D. & OETTGEN, H.F. (1973) Production of antibody to sheep red blood cells by human tonsil cells in vitro. Nature (Lond.), 243, 408. ISHIKAWA, T., WICHER, K. & ARBESMAN, E. (1972) Distribution of y (gamma) immunoglobulins in palatine and pharyngeal tonsils. Int. Arch. Allergy, 43, 801. JoNDAL, M., HOLM, G. & WIGZELL, H. (1972) Surfacemarkers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. ]. exp. Med. 136, 207. JONDAL, M., WIGZELL, H. & AIUTI, F. (1973) Human lymphocyte subpopulations: Classification according to surface markers and/or functional characteristics. Transplant. Rev. 16, 163. MISHELL, R.J. & DUTTON, R.W. (1967) Immunization of dissociated spleen cell cultures from normal mice. J. exp. Med. 126, 423. MOLLER, G. (1961) Demonstration of mouse isoantigen at the cellular level by fluorescence antibody technique. J. exp. Med. 114, 415. MOLLER, G. & PERSSON, U. (1974) Mitogenic properties of rabbit anti-human B2-microglobulin for murine B cells. Scand..3. Immunol. 3, 445. PLATTS-MILLS, T.A.E. & ISHIZAKA, K. (1975) IgG and IgA
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diphtheria antitoxin responses from human tonsil lymphocytes. J. Immunol. 114, 1058. RINGD&N, 0. & MOLLER, E. (1975) B cell mitogenic effects of rabbit anti-human B2 microglogulin for human lymphocytes. Scand. J. Immunol. 4, 171. RINGDEN, 0. (1976) Activation of human lymphocyte subpopulations by rabbit anti-human B2 microblobulin and by LPS. Scand. 5. Immunol. 5, (In press.) SAMARUT, C., BROCHIER, J. & REVILLARD, J.P. (1976) Distribution of cells binding erythrocyte-antibody (EA) complexes in human lymphoid populations. Scand. J. Immunol. 5, 221. SLOYER, J.L., VELTRI, R.W. & SPRINKLE, P.M. (1973) In vitro IgM antibody synthesis by human tonsil-derived lymphocytes. 5. Immunol. 111, 183. SMITH, R.S., SHERMAN, N.A. & NEWCOMB, R.W. (1974) Synthesis and secretion of immunoglobulin, including IgA, by human tonsil and adenoid tissue cultured in vitro. Int. Arch. Allergy, 46, 785. TABATA, T., KATSURAHARA, T., ENOMOTO, T., KITASHOJI, N. & TANAKA, S. (1975) Immunological function of human tonsil. Surface topology of human tonsil lymphocytes using the scanning electron microscope (SEM). Acta Otolaryngol. 80, 474. THORSBY, E. & BRATELI, A. (1972) A rapid method for preparation of pure lymphocyte suspensions. Histocompatibility Testing (ed. by J. Dausset and Colombani), p. 531. Munksgaard, A/S, Copenhagen. Transplant Rev. (1972) (ed. by G. Moller), Lymphocyte activation by mitogens. 11. Transplant. Rev. (1973) (ed. by G. Moller), T and B lymphocytes in humans. 16. WATANABE, T., YOSHIZAKI, K., YAGURA, T. & YAMAMURA, Y. (1974) In vitro antibody formation by human tonsil lymphocytes. J. exp. Med. 113, 608.
