Isolation and properties of the leukocytosis- and lymphocytosis-promoting factor of Bordetella pertussis.

Abstract

In a previous report on the effects of the leukocytosis-and lymphocytosispromoting factor (LPF) 1 ofBordetella pertussis on cyclic adenosine 3':5'-monophosphate (cAMP) metabolism, a method for isolation of highly active LPF was presented briefly (1). In the present communication, the results of further studies on the isolation and chemical, immunochemical, and biological properties of LPF are described. It has been found that a single moiety is responsible for the induction in mice of leukocytosis with both lymphocytosis and granulocytosis, histamine sensitization, and hypoglycemia and unresponsiveness to the hyperglycemic effects of epinephrine. In confirmation of our preliminary report (2), LPF has been found to be clearly distinct from the hemagglutinating pili of B. pertussis, the properties of which are also described. Materials and MethodsB. pertussis Cultures. Strain NIH 114 (3779B), which has been utilized in continuing studies in this laboratory was also employed in the present investigations. Lyophilizod samples, obtained through the courtesy of Dr. Charles Manclark, Bureau of Biologics, Bethesda, Md., were reconstituted in sterile distilled H~O and plated on Bordet-Gengou medium (Clinical Sciences Inc., Whippany, N. J.). The 2-day growth at 36°C on one Petri dish was harvested in 10 ml of 2% casamino acids (technical grade; Difco Laboratories, Detroit, Mich.), and 1 ml of the suspension was inoculated into 100 ml of liquid medium in i liter Blake bottles. After 2 days of incubation at 36°C, 10 ml of the seed culture (approximately 5 × 10 x° organJma) was inoculated into 1 liter of liquid medium in Povitsky bottles which were then incubated at 35-36°C. In order to provide maximum surface area for aeration, all bottles were placed in the horizontal position during incubation. In the case of the 100 ml cultures, the depth of the medium was 1.0 cm and the surface area 218 cm2; the values for the 1-1iter cultures were 2.5 cm and 580 cm 2, respectively.The liquid medium was that previously described (3) except for the omission of an anion exchange resin which was found to be unnecessary for either optimal growth, maintenance of the Phase I state, or maximal production of LPF. Culture density was assessed with the National Institutes of Health opacity standard, and the criteria utilized to determine whether the organisms were Phase I included: a homogenous population of small, gram-negative coccobacilli and

Polymorphonuclear Leukocyte‐Inhibitory Factor of Bordetella pertussis

“…This in vivo PIF activity, nevertheless, could not be separated from the histamine-sensitizing activity (HSF) by the DEAE chromatography. Therefore it may still be possible that the lymphocytosis (leukocytosis)-promoting factor which has been assumed to be identical with HSF (11,12) is responsible for both the blood leukocytosis and the observed failure of PMN to migrate from the blood vessels. Further investigation is necessary to ascertain the relationship between these factors and to clarify the mechanism of the PIF activity.…”

Human T lymphocyte mitogenesis in response to the B oligomer of pertussis toxin is associated with an early elevation in cytosolic calcium concentrations

“…in certain cell systems, the B oligomer may play a more active role in the modulation of cellular functions has been provided by Tamura et al [lo] who found that the resolved B oligomer of pertussis toxin stimulated mitosis in murine splenocytes and enhanced glucose oxidation in rat adipocytes. Despite the important implications of these findings with respect to the lymphocytosis-promoting [3,4] and hypoglycemic [2] physiological effects of the toxin, the mechanism of action of B-oligomer-mediated responses has remained undefined.…”

“…Strnad, Department of Pharmacology and Toxicology, Medical College of Virginia, Box 613 MCV Station, Richmond, VA 23298-0001, USA Pertussis toxin, produced by the bacterium Bordetella pertussis, first gained recognition for its ability to produce certain in vivo responses including sensitization to histamine [ 11, hypoglycemia [2], and a leukocytosis associated with elevated levels of circulating lymphocytes [3,4]. The toxin consists of six subunits [5], the largest (28 kDa) of which is a catalytic moiety (the A subunit), while the other five, ranging in molecular mass from 9.3 to 23 kDa, are associated to form a pentameric binding component (the B oligomer).…”

Increase in Intradermal Vascular Permeability Caused by Pertussis Toxin from Bordetella pertussis

“…Munoz et al (9) reported that vascular permeability in the striated muscle of mice is increased by injecting PT intravenously (iv) ; however the PF of PT was not studied. Therefore, we examined histological changes in the skin tissues at the PT injection sites and showed that the PF reaction is one of the biological activities of PT.A variety of methods for assaying PT are known (6,8,11,16), but some problems, such as lack of simplicity, inaccuracy, and high cost remain. We found that the use of PF of PT is a simple and accurate method to determine the biological activity of PT.…”

“…A variety of methods for assaying PT are known (6,8,11,16), but some problems, such as lack of simplicity, inaccuracy, and high cost remain. We found that the use of PF of PT is a simple and accurate method to determine the biological activity of PT.…”