Immunology, 1975, 28, 577.
C3 Receptors on Direct Plaque-forming Cells R. RAMASAMY AND HEATHER WILLIAMS Department of Pathology, Tennis Court Road, Cambridge
(Received 12th July 1974; accepted for publication 23rd July 1974) Summary. Using a rosette technique it is shown that only a small proportion of direct plaque-forming cells possess detectable C3 receptors 5 and 7 days after antigenic stimulation. The significance of this result is discussed. INTRODUCTION Direct plaque-forming cells (PFC) have been reported not to possess C3 receptors (Bianco, Patrick and Nussenzweig, 1971). We have used a rosette technique to visualize directly the presence of C3 receptors on direct PFC. Sheep erythrocytes coated with IgM and complement which are normally used as indicator cells to detect C3 receptors would lyse during the addition of guinea-pig complement to develop plaques. McConnel (1971) described the use of fowl erythrocytes which, on lysis, show a visible nucleus within a 'ghost' membrane. Fowl erythrocytes sensitized with optimal concentrations of IgM antibody and mouse complement were therefore used as indicator cells in these experiments.
MATERIALS AND METHODS Mouse anti-fowl erythrocyte serum Mice were injected with 10 per cent washed fowl erythrocytes in saline intraperitoneally and bled 7 days later. The IgM fraction was isolated as the exclusion peak by gel-filtration through a Sephadex G-200 column. This was then used to sensitize indicator cells at a sub-agglutinating dilution. Mouse complement Serum was obtained from the C5-deficient strains of mice DBA2 and A/J (Herzenberg, Tachibana, Herzenberg and Rosenberg, 1963). The complement was used undiluted.
Primed lymph node cells (LNC) Six to 10-week-old BALB/c mice were injected in their front foot pads with a 10 per cent solution of sheep erythrocytes. At different times after injection the axillary lymph nodes were removed and teased apart in Eagle's medium buffered with Hepes. The cells were washed once and resuspended at 5 x 106 cells per ml. C3 rosette formation Fowl erythrocytes were obtained by bleeding into acid-citrate-dextrose (ACD). The Correspondence: Dr R. Ramasamy, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge.
577
578 R. Ramasamy and Heather Williams cells were washed three times and sensitized with an IgM fraction of mouse anti-chicken erythrocyte serum at a subagglutinating dilution. The cells were then washed and treated for 15 minutes at 370 with undiluted mouse C5-deficient complement, which had been preabsorbed with chicken erythrocytes in the cold. The sensitized erythrocytes were washed and resuspended at 1 per cent v/v in Eagle's/Hepes medium. Fifty microlitres of LNC suspension was then mixed with 50 yul of the indicator cells in a narrow siliconed tube and centrifuged at 50-75 g in the cold. Plaque formation The rosetted pellet was gently resuspended in 0 05 ml of medium. 0 05 ml of a 10 per cent suspension of sheep erythrocytes was added together with 0-2 ml of agarose at 460 and the mixture rapidly plated on agar-coated slides. The slides were then developed for direct plaques as described by Jerne (Jerne, Nordin and Henry, 1963). The slides were fixed in a 1 per cent solution of formaldehyde in saline and maintained at 40 before scoring.
Scoring of rosetting plaque-forming cells (RPFC) A coverslip was gently overlaid on the agarose layer and plaques viewed under low magnification (x 40). If a lymphoid cell was clearly visible in the centre of the plaque, it was examined under higher power for rosetting erythrocytes. Three or more adherent erythrocytes were taken to indicate a positive reaction.
~~~~~.~~~. t~~~~~i ~ ~~
~~W
FIG. 1. A C3 rosette and direct plaque-forming cell under low magnification.
A rosette and plaque-forming cell (RPFC) is shown in Figs 1 and 2. At least eight replicate preparations of rosetted cells were plaqued and the individual counts were added together.
RESULTS The results of some experiments to determine the proportion of direct PFC which carry C3 receptors (RPFC) is summarized in Table 1.
579
C3 Receptors on Direct PFC
FIG. 2. The same C3 rosette and direct plaque-forming cell (RPFC) under high magnification. TABLE 1
C3
RECEPTORS ON DIRECT
PFC
Experiment 1 Proportion of C3+ LNC before plaquing Proportion of C3+ LNC after plaquing Proportion of LNG-forming rosettes with IgM-coated indicator cells Proportion of RPFC 5 days primed cells 7 days primed cells Total numbers of RPFC in four experiments Proportion of RPFC with 03-coated indicator cells (65/641) Proportion of RPFC with IgM-coated indicator cells (3/148)
29-5%
23-0% 0
164%
10.7% 10-1% 2-0% (P
C3 Receptors on Direct Plaque-forming Cells R. RAMASAMY AND HEATHER WILLIAMS Department of Pathology, Tennis Court Road, Cambridge
(Received 12th July 1974; accepted for publication 23rd July 1974) Summary. Using a rosette technique it is shown that only a small proportion of direct plaque-forming cells possess detectable C3 receptors 5 and 7 days after antigenic stimulation. The significance of this result is discussed. INTRODUCTION Direct plaque-forming cells (PFC) have been reported not to possess C3 receptors (Bianco, Patrick and Nussenzweig, 1971). We have used a rosette technique to visualize directly the presence of C3 receptors on direct PFC. Sheep erythrocytes coated with IgM and complement which are normally used as indicator cells to detect C3 receptors would lyse during the addition of guinea-pig complement to develop plaques. McConnel (1971) described the use of fowl erythrocytes which, on lysis, show a visible nucleus within a 'ghost' membrane. Fowl erythrocytes sensitized with optimal concentrations of IgM antibody and mouse complement were therefore used as indicator cells in these experiments.
MATERIALS AND METHODS Mouse anti-fowl erythrocyte serum Mice were injected with 10 per cent washed fowl erythrocytes in saline intraperitoneally and bled 7 days later. The IgM fraction was isolated as the exclusion peak by gel-filtration through a Sephadex G-200 column. This was then used to sensitize indicator cells at a sub-agglutinating dilution. Mouse complement Serum was obtained from the C5-deficient strains of mice DBA2 and A/J (Herzenberg, Tachibana, Herzenberg and Rosenberg, 1963). The complement was used undiluted.
Primed lymph node cells (LNC) Six to 10-week-old BALB/c mice were injected in their front foot pads with a 10 per cent solution of sheep erythrocytes. At different times after injection the axillary lymph nodes were removed and teased apart in Eagle's medium buffered with Hepes. The cells were washed once and resuspended at 5 x 106 cells per ml. C3 rosette formation Fowl erythrocytes were obtained by bleeding into acid-citrate-dextrose (ACD). The Correspondence: Dr R. Ramasamy, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge.
577
578 R. Ramasamy and Heather Williams cells were washed three times and sensitized with an IgM fraction of mouse anti-chicken erythrocyte serum at a subagglutinating dilution. The cells were then washed and treated for 15 minutes at 370 with undiluted mouse C5-deficient complement, which had been preabsorbed with chicken erythrocytes in the cold. The sensitized erythrocytes were washed and resuspended at 1 per cent v/v in Eagle's/Hepes medium. Fifty microlitres of LNC suspension was then mixed with 50 yul of the indicator cells in a narrow siliconed tube and centrifuged at 50-75 g in the cold. Plaque formation The rosetted pellet was gently resuspended in 0 05 ml of medium. 0 05 ml of a 10 per cent suspension of sheep erythrocytes was added together with 0-2 ml of agarose at 460 and the mixture rapidly plated on agar-coated slides. The slides were then developed for direct plaques as described by Jerne (Jerne, Nordin and Henry, 1963). The slides were fixed in a 1 per cent solution of formaldehyde in saline and maintained at 40 before scoring.
Scoring of rosetting plaque-forming cells (RPFC) A coverslip was gently overlaid on the agarose layer and plaques viewed under low magnification (x 40). If a lymphoid cell was clearly visible in the centre of the plaque, it was examined under higher power for rosetting erythrocytes. Three or more adherent erythrocytes were taken to indicate a positive reaction.
~~~~~.~~~. t~~~~~i ~ ~~
~~W
FIG. 1. A C3 rosette and direct plaque-forming cell under low magnification.
A rosette and plaque-forming cell (RPFC) is shown in Figs 1 and 2. At least eight replicate preparations of rosetted cells were plaqued and the individual counts were added together.
RESULTS The results of some experiments to determine the proportion of direct PFC which carry C3 receptors (RPFC) is summarized in Table 1.
579
C3 Receptors on Direct PFC
FIG. 2. The same C3 rosette and direct plaque-forming cell (RPFC) under high magnification. TABLE 1
C3
RECEPTORS ON DIRECT
PFC
Experiment 1 Proportion of C3+ LNC before plaquing Proportion of C3+ LNC after plaquing Proportion of LNG-forming rosettes with IgM-coated indicator cells Proportion of RPFC 5 days primed cells 7 days primed cells Total numbers of RPFC in four experiments Proportion of RPFC with 03-coated indicator cells (65/641) Proportion of RPFC with IgM-coated indicator cells (3/148)
29-5%
23-0% 0
164%
10.7% 10-1% 2-0% (P
