John J. Munoz

Acute experimental autoimmune encephalomyelitis in mice: I. Adjuvant action of Bordetella pertussis is due to vasoactive amine sensitization and increased vascular permeability of the central nervous system

Abstract Experimental autoimmune encephalomyelitis (EAE) in mice is dependent upon the use of Bordetella pertussis suspensions as an adjuvant. Intravenous administration of B. pertussis causes an increased vascular permeability in brain tissue and an increased vascular sensitivity to vasoactive amines which promotes the development of EAE. The efficacy of different batches and strains of B. pertussis in the expression of EAE closely correlates with the vasoactive amine sensitization activity of each material tested. Pertussigen, the histamine sensitizing factor (HSF), is responsible for these adjuvant properties whereas purified endotoxin is inactive. The effect of cimetidine, diphenhydramine, methysergide, reserpine, and cyproheptadine on B. pertussis induced histamine sensitivity and the expression of EAE are examined. Cyproheptadine, an agent with mixed histamine and serotonin blocking properties, blocks both B. pertussis-induced vasoactive amine sensitization and the expression of EAE.

Elicitation of experimental allergic encephalomyelitis (EAE) in mice with the aid of pertussigen.

Pertussigen, one of the biologically active proteins from Bordetella pertussis, was found highly active as an adjuvant to promote the induction of experimental allergic encephalomyelitis (EAE) in (SJL X BALB/c)F1 mice that had received at the same time an injection of mouse spinal cord (MSC) homogenized in complete Freunds adjuvant containing 4 mg of Mycobacterium tuberculosis H37RA per milliliter (CFA-H37). In this system 2 mg of MSC induced EAE, but a dose of 4 mg was more effective. As little as 250 ng of pertussigen facilitated induction of EAE, and 400 ng uniformly did so. Pertussigen was most effective when given iv from 1 day before to 5 days after administration of MSC homogenized in CFA-H37, when a uniform and severe disease was induced 11-13 days after immunization. Pertussigen given as late as 20 days after MSC-CFA-H37 still precipitated a mild form of EAE which appeared 8-12 days after the injection of pertussigen. When pertussigen was given 5 days after immunization, a chronic, nonfatal type of EAE was induced, and this persisted for the entire 74 days of observation. Histologic findings in the brain and spinal cord 15 days after sensitization in mice which received pertussigen and developed EAE showed perivascular infiltrates consisting mainly of mononuclear cells.

Action of pertussigen (pertussis toxin) on serum IgE and on Fcϵ receptors on lymphocytes

Abstract Pertussigen (pertussis toxin (PT)) is one of the most effective stimulators of IgE production in mice and rats. Employing flow microfluorimetric analysis (FMF), we showed that PT increases the percentage of blood and spleen lymphocytes with IgE on their surface. The percentage of IgE-bearing cells in the spleen of normal untreated C57B1/10SCN mice of various ages varied from 2.2 to 12.2%, with an average value of 6.1 ± 5.4%. In mice treated with 400 ng of PT and 1 mg of chicken egg albumin (EA), the percentage of these cells increased, 14 days after immunization, to an average value of 31.1 ± 2.2%. Immunization of mice with PT alone increase the percentage of IgE-bearing cells only slightly (13.1 ± 2.2% of the splenic lymphocytes) while injection of 1 mg of EA alone did not have any detectable action. As little as 6 ng of PT, when given simultaneously with 1 mg of EA, increased the percentage of IgE-bearing lymphocytes. A booster dose of 10 μg of EA given on Day 14 induced a further increase in the percentage of these cells even when as little as 0.039 ng of PT had been given at the time of initial immunization. PT was effective when given 4 days before or 5 days after EA. EA was effective when given 4 days before or 4 days after PT, but not 8 days after. The increase in IgE-bearing cells was mainly due to cytophilic binding of IgE to receptors for the ϵ chain of IgE (Fcϵ) on the surface of lymphocytes rather than to a greater number of IgE-producing cells. This was shown by removing the IgE from Fcϵ receptors by acid treatment which reduced the percentage of IgE-bearing cells to nearly normal values. The antibodies of IgE class with specificity to EA were increased dramatically, while antibodies with specificity to PT were not detected.

Adoptive transfer of experimental allergic encephalomyelitis in mice with the aid of pertussigen from Bordetella pertussis

Adoptive transfer of experimental allergic encephalomyelitis (EAE) in (SJL X BALB/c)F1 mice was accomplished by an iv injection of 2.4 to 4.7 X 10(7) lymph node cells (LNC) from mice immunized with mouse spinal cord emulsified in complete Freunds adjuvant when both donors and recipients had been treated iv with 400 ng of pertussigen at the time of immunization for the donors and on transfer of cells for the recipients. Pertussigen was essential in both donors and recipients for development of frank EAE. Signs of EAE in recipients were delayed, appearing 21-23 days after cell transfer; the maximum response at about Day 27 is considerably delayed in comparison with other reported studies on passive transfer of EAE. Histologically, recipient mice with paralysis due to EAE had typical perivascular infiltrates of mononuclear cells in the brain and spinal cord. The mechanisms by which pertussigen promotes the development of EAE after adoptive transfer of sensitized LNC are uncertain.

Role of Pertussigen (Pertussis Toxin) on the Mouse Protective Activity of Vaccines Made from Bordetella Species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 × 107 killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 μg of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild‐type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild‐type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non‐specifically to enhance the mouse protective action of those antigens.

Effect of Bordetella pertussis extract and vasoactive amines on vascular permeability

Vasxular permeability to Evans blue dye and 131-I-labeled human serum albumin was studied in normal mice and in mice treated with alkaline saline extracts (SE) from Bordetella pertussis cells. Skin sites inoculated intracutaneously with small doses of histamine, serotonin, or a combination of these 2 substances were more permeable in SE-treated mice than in normal animals. Intravenously administered catecholamines were able to reduce in varying degrees the vascular permeability induced by serotonin or by histamine in normal mice; in SE-treated mice the catecholamines were less effective. The relative effectiveness of intravenously administered catecholamines to reduce vascular permeability in normal or SE-treated mice was ranked as follows: isoproterenol greater than epinephrine greater than norepinephrine. When catecholamines were given concomitantly with histamine and serotonin in the skin test site, the permeability in both normal and SE-treated mice was again reduced or blocked, but isoproterenol was only weakly effective in this instance. Their relative effectiveness was epinephrine greater than norepinephrine greater than isoproterenol. The possible explanations for these results are discussed.

Effect of Dermonecrotic Toxin of Bordetella pertussis on the Spleen of CFW and C57BL/10ScN Mice

The dermonecrotic toxin (DNT), also called heat-labile toxin, was discovered by Bordet and Gengou (3) in extracts of Bordetella pertussis cells. The toxin had a marked necrotic effect on the skin of rabbits and guinea pigs and a degenerative action on the spleens of mice (5, 7, 12). The toxicity was inactivated at 56 C for 10 min (4,6,8). In this note we describe a pronounced difference in the response of spleens from CFW (random bred) and C57BL/IOScN (inbred) mice, raised in the animal production unit at the Rocky Mountain Laboratory, to the effects of this toxin. To our knowledge, differences among strains of mice in their response to purified DNT have not been recorded in the literature. DNT was prepared by a modification of Iida and Okonogis method (5) and partly Nakases method (9) from a Phase I Maeno strain of B. pertussis (agglutinogen type 1.2.3.4) grown in a modified synthetic medium of Stainer and Scholte (composition given in Ref. 1). The medium (500 ml per 4-liter Erlenmeyer flask) was inoculated with 2 ml of a cell suspension containing 25 X 109 cells per ml. The inoculum was prepared from 24-hr-old Bordet-Gengou agar cultures (composition given in Ref. 7). The inoculated flasks were incubated for 5 days at 35 C under stationary conditions; the cells were then collected by centrifugation (3,000 xg for 1 hr) and resuspended in cold sterile water to a concentration of 1012 cells per ml. These cells were sonically disrupted (kept cold with iced water) in a Biosonik III apparatus (Bronwill Scientific, Rochester, N.Y.) at 20 kHz for 5 min. The disrupted cells were centrifuged at 27,000 xg for 20 min to remove the cell debris, and the supernatant containing the DNT was collected by decantation. All steps were carried out at 2 to 10 C. About 6 ml of the clear solution of DNT was passed through a DEAE-Sephacel column (2.5 X 60 cm) equilibrated in 0.01 M phosphate buffer (pH 8.0) containing 20 mM sucrose. The column was successively eluted with (i) 800 ml of0.01 Mphosphate buffer, pH 8; (ii) 450 ml of 0.05 Mbuffer, pH 7.2; and (iii) 800 ml of 0.2 M buffer, pH 7.0. All the buffers contained 20 mx sucrose as a stabilizer of the toxin. The toxin, assayed by its dermonecrotic action in the

PURIFICATION AND ACTIVITIES OF PERTUSSIGEN, A SUBSTANCE FROM BORDETELLA PERTUSSIS

pertussis is described. Starting from acetone extracted cells, pertussigen was solubilized by 1 M sodium chloride in 0.05 M sodium pyrophosphate at pH 7.4. The extract was then fractionated by a combination of hydroxylapatite column chromatography, precipitation at pH 4.5 and sucrose density gradient electrophoresis. All fractions and the original cells were tested in mice for their ability to increase susceptibility to histamine, induce lymphocytosis, stimulate IgE with specificity to hensT egg albumin, induce hypoglycemia, and protect mice from intracranial infection with B. pertussis. In addition, these fractions were tested for their ability to enhance the susceptibility to experimental allergic encephalomyelitis in Lewis rats. All of these activities were found in the purest preparation of pertussigen, and it is thus thought that they are all induced by this substance.