Immunology 1977 32 743

Inhibition of antibody responses by cells from mice treated with picryl sulphonic acid W. R. THO MAS & G. L. ASHERSON Division of Immunology, Clinical Research Centre, Watford Road, Harrow, Middlesex

Received 17 August 1976; accepted for publication 24 September 1976

Summary. Cells from mice inoculated with picryl sulphonic acid (PSA cells), which contain suppressor T cells for contact sensitivity to picryl chloride, were examined for their ability to alter antibody responses of normal mice. These cells did not influence antibody or plaque-forming cell (PFC) production accompanying contact sensitivity reactions produced by painting with picryl chloride but reduced IgG antibody and indirect PFC responses to conjugates of trinitrophenyl (TNP) bovine serum albumin and ovalbumin. IgG responses to TNP or new antigenic determinants of TNP-mouse serum albumin were not affected by PSA cells. The PSA cells required several weeks to produce reductions of responses and only reduced responses to optimal doses of antigen. When the injection of antigen was delayed until several weeks after the injection of PSA cells rapid reductions of responses were found but these were short-lived. The inhibition was specific for TNP proteins although responses to hapten and carrier were reduced. Evidence is presented to show that the inhibition was mediated by an adherent macrophage-like cell rather than a T cell. The inhibitory activity was resistant to irradiation and anti-C treatment but was removed by glutaraldehyde treatment and cotton wool filtration.

INTRODUCTION Mice injected i.v. with picryl sulphonic acid (PSA) become unresponsive to immunization with immunogenic trinitrophenyl (TNP) derivatives. The unresponsiveness involves both antibody production (Fidler & Golub, 1973; Elson & Taylor, 1974) and contact sensitivity (Asherson, Zembala & Barnes, 1971). Since injection of PSA produces suppressor T cells which inhibit contact sensitivity reactions to picryl chloride (Zembala & Asherson, 1973) it was of interest to see if the suppressor cells could also influence antibody responses to TNP derivatives. Accordingly mice were injected with PSA in a regime shown to produce suppressor T cells and the ability of these cells to influence antibody responses of mice to picryl chloride and trinitrophenylated proteins examined. The results show that cells from mice treated with PSA did not alter the antibody response which accompanies contact sensitivity reactions to picryl chloride. However, some inhibition of responses to TNP-proteins was observed. Evidence is presented to show the inhibition was mediated by a macrophagelike cell rather than a T cell even though the inhibition was specific for TNP-proteins. The ability of these cells to inhibit immune reactions was apparently restricted to conditions involving strong antigenic stimulation.

Correspondence: Dr W. R. Thomas, Division of Immunology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ.



W. R. Thomas & G. L. Asherson

MATERIALS AND METHODS Mice 6-12 week-old male mice bred at the Clinical Research Centre were used.

Preparation of trinitrophenyl-protein conjugates (TNPproteins) Bovine serum albumin (Cohn V fraction) (Miles Laboratories Ltd, Stoke Poges) (BSA), egg albumin (BDH, Poole, Dorset) (OA) and mouse serum albumin (MSA) were conjugated with picryl sulphonic acid by a slight modification of the method of Rittenberg & Amkraut (1966). Mouse serum albumin was prepared using the TCA method described by Rubin (1973) and was fractionated through Sephadex G-100 to remove aggregates. The TNP-BSA, TNP-MSA and TNP-OA had an average of approximately 10 TNP groups per protein molecule. Immunization procedures Sensitization with picryl chloride. Picryl chloride (BDH, Poole, Dorset) supplied moist was made to a nominal 5 per cent in ethanol just prior to use. 0 1 ml of this solution was applied to the shaven thorax and abdomen of mice. Administration of picryl sulphonic acid. Picryl sulphonic acid (Sigma, St Louis, Missouri) (PSA) was dissolved in PBS and the solution adjusted to neutral pH with concentrated sodium hydroxide. 5 mg of PSA was given intravenously per mouse. Immunization ofprotein (and TNP-proteins). Equal volumes of protein solution and Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan) (FCA) were emulsified and a total of 20 p1 (usually containing 200 ug of antigen) injected into six s.c. sites. 20 p1 were given in each hind footpad and 40 ,ul were given to each of the four dorsal sites. Cell preparation and transfer Lymph nodes (usually inguinal, axillary, subscapular and mesenteric) were dissociated by pressing through a wire grid and then washed. Generally 3 x 10' viable cells (as judged by trypan blue exclusion) were injected i.v. into recipients.

Antibody titrations Haemagglutination. Preparation of indicator cells.

Proteins (BSA, OA, TNP-BSA, TNP-OA, TNPMSA) were coupled to washed sheep erythrocytes (SRC) with glutaraldehyde by the methods of Rubin (1973). TNP-coated SRC were prepared by reacting PSA cells with SRC by the method of Rittenberg & Pratt (1969) and then incubating 12 5 per cent washed TNP-SRC (in PBS) with 0 66 per cent glutaraldehyde for 30 min at room temperature. Assay procedure. 0 2 ml of serum was absorbed with 01 ml of packed glutaraldehyde-treated SRC (prepared by incubating a 50 per cent suspension of SRC in 1 per cent glutaraldehyde for 30 min at room temperature). This procedure was shown to remove the reactivity ofnormal CBA serum to glutaraldehydetreated indicator cells. Assays were performed using the glutaraldehyde-treated indicator cells and PBS with 1 per cent normal rabbit serum as diluent. Cooke microtitration apparatus was used (Cooke Engineering Co., Alexandria, Virginia) and 0 025 ml of 0 5 per cent indicator cells was added to the same volume of diluted serum. Titres were read after incubation for 60 min at 370 and overnight at room temperature and recorded as the highest dilution showing agglutination. The results have been expressed as log2 titre. Any value less than 1/2 (titre = 1) has been designated as 0. Assay of new antigenic determinants (NAD). (Method of Rubin, 1973). The NAD of TNP-MSA were assayed by titrating sera against TNP-MSA coated indicator cells with 10-3 M e-mono-TNP-Llysine in the diluent [prepared by the method of Okymura & Satake (1960)]. The TNP-lysine was shown to be ten-fold in excess of the concentration required to completely inhibit the reactivity of anti TNP-MSA to TNP-BSA-coated indicator cells. The titre against TNP-MSA coated cells in the presence of TNP lysine has been called the anti NAD titre. The anti TNP titres of the same sera were assayed against TNP-OA coated indicator cells. Mercaptoethanol treatment. 0 025 ml of serum was incubated with 0-025 ml of 0 2 M 2-mercaptoethanol (BDH, Poole, Dorset) in the microtitration plates for 30 min at 37°. The plates were then left uncovered on the bench for 2 h at room temperature to help remove excess mercaptoethanol before titration. The mercaptoethanol-sensitive titre has been expressed as the difference in log2 titres of titration with and without mercaptoethanol. Mercaptoethanol resistant antibodies will be referred to as IgG and mercaptoethanol sensitive as IgM.


Inhibition of antibody responses Haemolytic antibody

5 mg PSA and after 4 days removing their limb girdle and mesenteric lymph nodes. Cell suspensions prepared from these nodes (PSA cells) were transferred to normal recipients (usually 3 x 107 cells/mouse) i.v. It was found that PSA injection did not elicit antibody, detectable 3, 10, 21 or 31 days after inoculation, but that injection of PSA cells into normal recipients induced low titres (1-2) of IgM (mercaptoethanol sensitive) antibody.

Anti-TNP haemolytic antibody was titrated with unfixed TNP-SRC. To 0025 ml dilutions of serum diluted in microtitre plates were added 0-025 ml of guinea-pig complement and 1 per cent TNP-SRC. The titre was the highest dilution showing complete lysis after incubation for 1 h at 37°.

Plaque-forming cell determination PFC were determined using a slightly modified version of the Cunningham-Szenberg (1968) technique. TNP-SRC were used as target cells (Rittenberg & Pratt, 1969). Indirect PFC were determined by adding rabbit anti mouse globulin to the assays. The antiserum was titrated to find the concentration developing the maximum number of indirect PFC.

Failure of PSA cells to affect antibody production accompanying contact sensitivity to picryl chloride Mice were injected with 3 x 107 PSA cells and painted with 0-1 ml of 5 per cent picryl chloride, a regime previously used to demonstrate suppressor activity of PSA cells on contact sensitivity. After 6 days the mice were challenged with the original dose of picryl chloride. Antibody titres developing 2 and 7 days after this challenge, as well as the titres of the primary response, were determined. Four similar experiments were performed using 3 x 107_108 cells/ mouse. As shown for one experiment (Fig. 1) PSA cells did not alter primary IgM titres or the IgM and IgG (mercaptoethanol resistant) titres which followed a second painting. Plaque-forming cells (PFC) to TNP can be detected in the spleen 5 days after painting with picryl chloride.

Anti-C treatment The procedures for preparation and use of anti-C and complement have been described in some detail (Asherson, Allwood & Mayhew, 1973). Treatment of lymph node cells with anti-C and complement destroyed 60-70 per cent of the population.

Cotton wool absorption The method used for removal of adherent cells was that of Janossy & Greaves (1971), shown to remove phagocytic cells. 108 cells suspended in Eagle's medium with 20 per cent foetal calf serum were incubated in a syringe containing 150 mg of absorbant cotton wool for 30 min at 370 and were then eluted with 6 ml of warm medium. This procedure permitted a 65 per cent recovery of cells in the eluate.

anti-TNP 9 Picryl chloride

Picryl chloride

chal lenge

cells 8[PSA I 7_




Irradiation 6 Cells were irradiated in vitro from a 59Co source atT approximately 1-5 rad/s.

Glutaraldehyde treatment Cells (1081/ml) were incubated in a solution of 0 5 per cent glutaraldehyde for 1 h at room temperature before washing.



4 3 2


I 2

3 4




7 8 Days

9 10 11 12 13

Cell population transferred (PSA cells) The following experiments examine the effect of cells shown to suppress contact sensitivity reactions (Zembala & Asherson, 1973) on antibody responses. These cells were prepared by injecting mice i.v. with

Figure 1. Effect of PSA cells on responses to picryl chloride. Mice were given 3 x 107 PSA or normal cells on day 0 and painted with picryl chloride on day 0 and 6. Each result is the mean + s.d. of titres of groups of five mice. Groups were given PSA cells (P), normal cells (N) or no cells (C). (-) IgG; (D) IgM. No significant difference of titre produced by PSA cells was observed.

W. R. Thomas & G. L. Asherson

746 anti -TNP


6-J 4 3 2

20 3 D



31 42 52 Days Figure 2. Effect of PSA cells on anti TNP response to TNPBSA. Each point represents mean + s.d. of groups of five mice given PSA cells (0), normal cells (z) or no cells (a). Mice were given 3 x 107 PSA or normal cells and immunized with 200 pug TNP-BSA on day 0. The titres of PSA-cell-treated mice were less than with the control groups on day 42 and 53 ) IgM; ( ) IgG. (P

Inhibition of antibody responses by cells from mice treated with picryl sulphonic acid.

Immunology 1977 32 743 Inhibition of antibody responses by cells from mice treated with picryl sulphonic acid W. R. THO MAS & G. L. ASHERSON Division...
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