Bourke R. Maddox and David W. Scott

Immunology Department, American Red Cross Holland Laboratory, Rockville, MD 20855 USA

Received 03/29/96; Accepted 06/10/96; On-line 06/25/96

Animals.

All transgenic animals used are lines previously described (4). Briefly, line 6.1 VH12 transgenic and non-transgenic littermates are identical to C57BL/10.A at the MHC locus (H-2^a), but carry the IgMb locus of the C57Bl/6 background. Ninety six to one hundred percent of splenic B cells from this line expressed the transgenic V_H12 idiotype (Id) found in the CH27 lymphoma (4, 6), and significant serum levels of Id were present by 4 weeks of age; ~66-75% of their B cells were CD5⁺. Anti-MHC class 1 transgenic mice (3.83; ref. 7), which possess rearranged IgM and IgD receptors specific for mouse H-2K^k, were originally provided by Dr. David Nemazee (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO). Transgenic and non-transgenic, normal littermates were obtained through backcrossing with BALB/cByJ from the Jackson Laboratory as previously published. The Sp6 (µ + kappa) anti-trinitrophenyl (anti-TNP) homo-zygous transgenic (Sp6; ref. 8) mice were generously provided by Dr. Rinus Lamers and the late Prof. Georges Köhler (Max Planck Institute for Immuno-biology, Freiburg, Germany) and have been back-crossed to C57Bl/6 or Balb/c.

Antibodies and other reagents.

Goat anti-mouse IgM, µ heavy chain-specific (Southern Biotech Associates, Inc. Birming-ham, AL), was used for all apoptosis and proliferation assays. Cells were cultured in RPMI-1640 (Bio-Whittaker, Walkersville, MD) with 5% heat inac-tivated fetal bovine serum (FBS, Sigma Chemical Company, St. Louis, MO) and 5 x 10^-5 M 2-mercaptoethanol. Lipopolysaccharide (LPS) from Escherichia coli 055:B5 and pokeweed mitogen (PWM) were both purchased from Sigma.

Cell preparation, proliferation and apoptosis assays.

Splenic single cell suspensions were obtained and red cells removed as described previously (3-5). Viable cell counts were performed using trypan blue exclusion, and suspensions were normalized to 5 x 10⁶ cells per ml. Unless otherwise noted, all spleens were processed individually, in parallel, on a given day.

Proliferation assays were performed as described earlier (5). Briefly, cells were cultured in 96 well tissue culture plates at 37°C, 7% CO₂, at 5x10⁵ cells per well in 200 ml complete medium. The indicated doses of anti-IgM (LPS or PWM; not shown) were added at time zero, and the cells pulsed at 44-50 hours with 0.5µCi [methyl-³H]-thymidine (Amersham Life Sciences, Inc.); plates were harvested at 50 hours on a Filtermate 196 harvester (Packard Instrument Company, Downers Grove, IL) onto glass fiber filters. Filters were allowed to air dry overnight and then read on a Matrix 9600 Direct Beta Counter (Packard).

For apoptosis (9-11), spleen cells were cultured for 24 hours at 37°C, 7% CO₂, in 24 well tissue culture plates. Doses of anti-IgM, as indicated, were added at time zero to one ml of 5 x 10⁶ cells/ml per well. Pellets were washed twice with cold PBS and then resuspended in 70% ethanol and maintained at 0°C for a minimum of one hour to fix. When noted, B cells were labeled with FITC-anti-B220 before fixation and the positive cells gated in order to only analyze B220⁺, IgM⁺ populations. After fixation, cells were then washed twice with cold PBS and resuspended in one ml PBS containing 10µg/ml RNase (Sigma) for 30 minutes at 37°C. Propidium iodide was added to a final concentration of 50µg/ml, and allowed to equilibrate for ten minutes. Samples were the analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA), using FL2 channel and low flow rate. FL2 area and width were used to gate out doublets, and front scatter and FL2 height were used to gate out debris; a minimum of 10,000 gated cells were collected for each sample. Final data was prepared using gated cells on an FL2 height histogram, with apoptotic cells appearing as a sub 2N population to the left of G₀/G₁ peak (9).

It should be noted that the numbers of B cells in each line was relatively constant, ranging from 38-65%, although the 6.1 line possessed a large number of B220^low cells, typical of the CD5 subset.