Difference in requirement of macrophage products for concanavalin A-induced production of eosinophil chemotactic factor and macrophage chemotactic factor from guinea pig lymphocytes in vitro

Differences between the conditions for an eosinophil chemotactic factor (ECF) and macrophage chemotactic factor (MCF) production by lymphoid cells of mesenteric lymph nodes and spleen were studied in guinea pigs. If lymphoid cells were washed less than 4 hr after concanavalin A (Con A) stimulation and were cultured for an additional 24 hr, they failed to produce ECF, whereas Con A stimulation for 1 hr before washing was sufficient to stimulate them to produce MCF. Subsequently, it was shown that heat-labile soluble factors (termed ECF-PF) with potentiating activity for ECF production are produced from macrophages by 5 micrograms/ml Con A activation. When ECF-PF were added to the cell culture with 5 micrograms/ml Con A, the lymphoid cells could produce ECF even when they were washed 2 hr after Con A stimulation and were cultured for an additional 24 hr, suggesting that ECF-PF plays a critical role in the early stage of ECF production. The lymphoid cells were also able to produce ECF even when they were cultured with ECF-PF and a suboptimal dose of Con A (1 microgram/ml) for ECF production. Protein synthesis seemed to be essential for ECF-PF production. The ECF-PF activity was associated with two separated molecular fractions with m.w. of about 50,000 to 70,000 and of 10,000 to 20,000. It is thus suggested that ECF is produced from T cells by Con A stimulation under conditions which differ, at least, from those for MCF in the requirement of ECF-PF.     

The appearance of eosinophil-directed chemotactic inhibitory factor in the serum of complete Freund's adjuvant-treated guinea pigs

When guinea pigs were treated by complete Freund's adjuvant (CFA), eosinophil-directed chemotactic inhibitory factor (ECIF) appeared in the serum of the treated animals. The inhibition of the response of eosinophils by ECIF was selective for the T cell-derived chemotactic factor, which has been isolated from DNP-ascaris extract-induced skin lesions (termed delayed ECF-a) but not for another ECF (termed delayed ECF-b), which was also isolated from the same skin lesions. ECIF was detected in the serum after a single CFA injection, and no significant increase of ECIF activity was observed in the serum of animals injected with CFA twice. The ECIF activity in the serum was associated with fractions of MW 70,000 and 12,500, and failed to bind to Con A-Sepharose. Furthermore, ECIF was absorbed by eosinophils but not by macrophages suggesting that eosinophils have receptor sites for ECIF. On the other hand, chemotactic inhibitor against delayed ECF-b was not detected in the serum. It was thus suggested that inhibition of eosinophil reaction by CFA treatment is related to ECIF, inhibition being selective for the eosinophil response to delayed ECF-a.     

Eosinophil and neutrophil chemotactic activities of adult worm extracts of Schistosoma japonicum in vivo and in vitro

Large numbers of eosinophils and neutrophils attracted to the soluble extract of Schistosoma japonicum adult worms (SjAW-ext) were detected at the injection site of normal guinea pig skin. Eosinophil and neutrophil chemotactic activities were also confirmed in in vitro assay by using blind-well chambers with Millipore filters in dose-dependent fashion. Two components of SjAW-ext showed eosinophil chemotactic activity; one was in the high molecular weight fraction (JAE-H), estimated to be more than 440,000 daltons, the other in the low molecular weight fraction (JAE-L) obtained by Sephadex G-200 gel filtration. High neutrophil chemotactic activity was detected in the JAE-L. These eosinophil and neutrophil chemotactic activities were also detected in culture fluid of S. japonicum adult worms. Eosinophil chemotactic factor (ECF) of JAE-H was stable to heating (100 C, 30 min) and pronase digestion, but completely destroyed by periodate oxidation. It is suggested that the ECF of JAE-H is a glycoprotein. JAE-L was also stable to heating (56 and 100 C, 30 min) and pronase digestion for eosinophil chemotaxis. Possible roles of those activities in schistosome infections are discussed.     

Modulation of the biologic activities of IgE-binding factor. II. Physicochemical properties and cell sources of glycosylation-enhancing factor

T lymphocytes of rats treated with Bordetella pertussis vaccine (BP) formed a soluble factor that enhanced the glycosylation of IgE-binding factors during their biosynthesis, and provided the latter factors with the biologic activity to potentiate the IgE response. The present experiments demonstrated that pertussigen (leukocytosis-promoting factor) from BP induced normal rat spleen cells to form the glycosylation-enhancing factor. The same factor was obtained by incubation of normal spleen cells with 5 micrograms/ml, but not 2 micrograms/ml, concanavalin A. When normal rat mesenteric lymph node cells were incubated with the glycosylation-enhancing factor together with IgE, IgE-binding factors formed by the cells selectively potentiated the IgE response. The IgE-binding factors formed by the same cells upon incubation with IgE alone neither enhanced nor suppressed the IgE response. The glycosylation-enhancing factor changed the nature of IgE-binding factors formed by the rat-mouse T cell hybridoma, 23A4. IgE-binding factors induced by IgE alone lacked affinity for lentil lectin, whereas those induced by IgE in the presence of the glycosylation-enhancing factor had affinity for the lectin. The cell source of the glycosylation-enhancing factor appeared to be W 3/25+ Fc gamma R+ T cells. The glycosylation-enhancing factor was protein in nature and had a m.w. of about 25,000. The factor had affinity for acid-treated Sepharose and could be recovered from the beads by elution with lactose. The factor was different from interleukin 2 with respect to both its affinity for galactose and its isoelectric point.     

Pertussigen in vivo enhances antigen-specific production in vitro of lymphokine that stimulates macrophage procoagulant activity and plasminogen activator

Pertussigen is a protein toxin of Bordetella pertussis that acts as a powerful stimulator of the intensity and duration of delayed-type hypersensitivity (DTH) in mice. This study describes the potent in vivo effect of pertussigen on the levels of antigen-specific macrophage-activating lymphokine(s); lymphokine(s) was measured by the stimulation of macrophage procoagulant activity (mPCA), or plasminogen activator (PA) activity. Lymphoid cells were removed from immunized animals and cultured with specific antigen, keyhole limpet hemocyanin, ovalbumin, or human gamma-globulin. The culture supernatants were then incubated with the monocyte-like cell line WEHI-265 to measure mPCA or with WEHI-265 or resident peritoneal macrophages to measure PA activity. Mice were given pertussigen at the time of immunization, and the subsequent generation by lymphocyte supernatants of both of these macrophage activities proved to be greatly enhanced; the effect of pertussigen was antigen specific. Pertussigen thus induces an increase in lymphokine(s) production responsible for the in vitro increase in macrophage mPCA and PA activity and which may be responsible for some of the potent immune effects of this agent in vivo.     

Cellular transfer of experimental allergic encephalomyelitis in Lewis rats: Effects of different sensitization regimens on in vitro reactivity of donor spleen cells to concanavalin A

Splenocytes from Lewis rats sensitized to guinea pig spinal cord (GPSC) and complete Freund's adjuvant (CFA) or to myelin basic protein (MBP)-CFA plus pertussis vaccine were less effective than spleen cells from MBP-CFA sensitized donors in transferring EAE to syngeneic recipients following culture with concanavalin A (Con A). Moreover, splenocytes from rats sensitized to GPSC-CFA plus pertussis vaccine showed no EAE transfer activity following culture with Con A. Diminished EAE transfer activity occurred in parallel with decreased proliferative responses of primed splenocytes to Con A. These effects were due, at least in part, to the use of pertussis vaccine and to Con A activation of a suppressive adherent cell subpopulation in sensitized donor spleens. Proliferative responses and EAE transfer activity were restored upon removal of plastic-adherent cells from splenocytes of rats sensitized to MBP-CFA plus pertussis vaccine prior to Con A activation of the non adherent lymphoid cells. Deletion of plastic-adherent cells from splenocytes of donors sensitized to GPSC-CFA plus pertussis vaccine prior to activation with Con A, however, had no effect on proliferative responses or EAE transfer activity. Furthermore, EAE transfer activity of Con A-activated splenocytes from MBP-CFA-sensitized donors was lost when such cells were cultured with splenocytes from donors sensitized to GPSC-CFA plus pertussis vaccine.     

Eosinophils and immune mechanisms. III. Production of the lymphokine eosinophil stimulation promoter by mouse T lymphocytes.

The lymphoid cell population responsible for production of eosinophil stimulation promoter (ESP), a lymphokine which increases migration of eosinophils, was investigated in murine Schistosoma mansoni infection. Con A challenge induced ESP production, whereas LPS did not. Prior treatment with anti-thetaC3H alloantiserum plus complement in vitro eliminated ESP production; in vivo treatment with rabbit anti-mouse thymocyte serum consistently reduced ESP production by splenic lymphoid cells, but affected lymph node cell ESP production only after exceptionally large doses. Thymocytes did not produce significant amounts of ESP; nor did lymphoid cells from congenitally athymic mice. Depletion of B lymphocytes and macrophages by nylon fiber adherence eliminated antigen-induced ESP production; this was partially restored by addition of non-immune, 72-hr peritoneal exudate cells. Con A-induced ESP production was not affected by nylon fiber treatment. These results demonstrate that ESP is produced by an ATS-sensitive, peripheralized T lymphocyte population, and suggest a macrophage requirement for antigen-induced production of this lymphokine.     

Studies on the adjuvant effect of Bordetella pertussis vaccine to sheep erythrocytes in the mouse. I. In vitro enhancement of antibody formation with normal spleen cells.

The adjuvant effect of Bordetella pertussis vaccine (PV) on the antibody response to sheep erythrocytes (SRBC) has been studied in vitro with the Mishell-Dutton immunization technique. The addition of PV to cultures of spleen cells obtained from normal non-immunized mice markedly enhanced the plaque-forming cell response to SRBC. The greatest enhancement was evident at 24 hr of culture. PV was also shown to enhance the antibody response of spleen cells that had been depleted of either T lymphocytes or adherent cells, presumably macrophages. In addition, it was found that PV, per se, released into the culture medium a soluble cell-free component(s) that contributed significantly to adjuvanticity. The results suggest that at least one of the ways that PV enhances the in vitro immune response to SRBC is by direct stimulation of precursors of antibody-forming cells.     

Suppression of T lymphocyte chemotactic factor production by the opioid peptides beta-endorphin and met-enkephalin

The opioid peptides beta-endorphin and met-enkephalin have been shown to modulate human lymphocyte proliferation, mononuclear cell locomotion, natural killer cell activity, and neutrophil locomotion. This study demonstrates that beta-endorphin and met-enkephalin inhibit the production of a T lymphocyte chemotactic factor (LCF) by concanavalin A (Con A)-stimulated peripheral blood mononuclear cells. Inhibition of LCF production was observed by using concentrations of 10(-11) to 10(-6) M beta-endorphin or met-enkephalin but not alpha-endorphin. A bimodal pattern of suppression of LCF production was observed with both met-enkephalin and beta-endorphin when titrated from 10(-12) to 10(-6) M concentrations, with the peaks of suppressive activity occurring at concentrations of 10(-11) M and 10(-6) M. Timed studies of the production of LCF over a 54-hr period showed that there was an appreciable lag in the onset of measurable LCF activity in mononuclear supernatants produced in the presence of beta-endorphin and met-enkephalin. The suppression of LCF production mediated by opioid peptides in mononuclear supernatants was abrogated by depletion of glass-adherent mononuclear cells before culturing with opioids and Con A. The inhibitory effect of opioid peptides on LCF production was prevented by the addition of indomethacin to cell cultures. Additional experiments showed that exogenous prostaglandin E2 (PGE2) suppressed Con A-stimulated LCF production when added at concentrations ranging from 10(-6) to 10(-8) M. Other studies suggested that the mechanism of opioid peptide-mediated suppression of LCF production was due to an enhanced sensitivity of mononuclear cells to the inhibitory action of PGE2. These data provide further evidence for modulation of the immune response in humans by the neuroendocrine hormones beta-endorphin and met-enkephalin and further suggest a link between this modulation and arachidonic acid metabolism.     

The formation of a test system for assessing the safety of different types of pertussis preparations: their cytotoxic action on mouse thymocytes in vitro

The test for the evaluation of the toxicity of different types of pertussis preparations as manifested by their in vitro influence on mouse thymic cells (T test) has been finally worked out. The use of the T test has made it possible to reveal the nonstandard character of the production lots of adsorbed diphtheria-pertussis-tetanus vaccines, both whole-cell vaccine and Japanese acellular vaccine. The degree of the in vitro damaging action of pertussis preparations on mouse thymic cells greatly depends on the residual content of Bordetella pertussis nontoxoidized toxin which, in contrast to B. pertussis lipopolysaccharide and filamentous hemagglutinin, produces pronounced cytotoxic action on mouse thymic cells.     

Characterization of human chemotactic lymphokine production induced by mitogens and mixed leukocyte reactions using a new microassay.

Quantification of lymphokine production in vitro can be a useful tool in the evaluation of delayed hypersensitivity in various disease states. A micro-method for the measurement of chemotactic lymphokine production by human mononuclear leukocytes (MNL's) has been developed. MNL's are isolated on Ficoll-Hypaque gradients and cultured without plasma in microtiter plates. Culture supernatants are harvested through glass fibre filter paper under vacuum in a semi-automatic harvester. Chemotactic lymphokine activity in the supernatants is quantified in miniaturized Boyden chambers using human monocytes as responder cells. The production of chemotactic activity can be initiated by mixed leukocyte reactions as well as by soluble antigens or mitogens, and therefore may be a useful adjunct in tissue typing. Studies of lymphokine production in normal individuals indicate that these methods are quantitative, reproducible, and readily applicable to the study of this parameter of immune function in human disease.     

Production of a human T lymphocyte chemotactic factor by T cell subpopulations

Concanavalin A-stimulated human peripheral blood mononuclear cells release a lymphocyte chemotactic factor. This lymphocyte chemotactic factor is produced optimally after 24 to 48 hr of culture and is not found before 3 hr of culture, which suggests that the factor is synthesized de novo and is not preformed and secreted after Con A stimulation. This is further supported by experiments showing that the protein synthesis inhibitors cycloheximide and puromycin totally prevent the production of the chemotactic factor. Experiments using cultured and uncultured T lymphocytes as responding cells show that cultured T cells respond more efficiently than uncultured T cells to this factor. Furthermore, the lymphocyte chemotactic factor preferentially stimulates T lymphocyte locomotion as compared to peripheral blood non-T lymphocyte migration. Fractionation of mononuclear cells into glass nonadherent lymphocytes, monocyte-enriched preparations, T lymphocytes, and non-T lymphocytes shows that lymphocyte chemotactic factor is produced by Con A-stimulated, glass nonadherent lymphocytes and T cells but not by monocytes or non-T lymphocytes. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T cell subpopulations shows that the production of T lymphocyte chemotactic factor can be attributed to the Leu-2 suppressor/cytotoxic T cell subset. The generation of a T lymphocyte chemotactic factor by Leu-2 T cells may represent a means of recruiting other T cells to the site of its release.     

Leukocyte accumulation in sparganosis: further characterization of an eosinophil chemotactic factor of the plerocercoid of Spirometra erinacei

An eosinophil chemotactic (ECF) was partially purified from plerocercoids of Spirometra erinacei by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. The molecular weight of ECF was estimated to be 25,000-45,000 by high-pressure liquid chromatography. The ECF was bound with concanavalin A-Sepharose. The ECF was sensitive to periodate oxidation and to heating (56 degrees C, 30 min). On isoelectric focusing, eosinophil chemotactic activity was clearly revealed at pI 4.1. These results suggest that ECF of S. erinacei plerocercoid is an acidic glycoprotein. An intradermal injection of ECF eosinophil attractions in the normal guinea pig skin.     

Characteristics of the immune response to a single peroral administration of different doses of corpuscular pertussis vaccine

The effect of oral administrations of different doses of pertussis vaccine on the humoral and cell-mediated responses of systemic immunity and on the immunomorphological transformation of the mucous membrane of the small intestine was studied in (CBA X C57BL/6)F1 mice. On day 28 after the administration of all the tested doses of pertussis vaccine the animals were found to have a high degree of protection from the development of meningoencephalitis induced by the inoculation of Bordetella pertussis in the absence of specific hemagglutinins in their blood sera. At the same time the formation of spontaneous and immune rosette-forming cells and splenocytes was found to be inversely related to the administered dose. The immunomorphological transformation observed in the mucous membrane of the small intestine and in the lymphoid tissue associated with the small intestine was indicative of the stimulation of local immunity. The results thus obtained suggest that a single oral administration of pertussis vaccine to mice stimulates cell-mediated and humoral reactions of local immunity and induces the development of systemic cell-mediated immunity.     

Review of the biology of Bordetella pertussis.

Bordetella pertussis produces a complex array of adhesins, aggressins and toxins that are presumed to be important in the colonisation of its human host and in ensuring its survival and propagation. The organism also has highly sophisticated mechanisms for regulating virulence factor expression, in response to environmental signals or by reversible mutations. Despite the rapidly increasing knowledge of these aspects of the biology of B. pertussis, our understanding of the pathogenesis of whooping cough is still far from clear. In defining the role of individual factors, reliance has to be placed on in vitro assays or animal models of the human infection, particularly in the mouse, where different conditions may prevail. Some clues to pathogenic mechanisms may be provided by considering other bordetellae, especially B. parapertussis, B. bronchiseptica and B. avium, their similar, but not identical, range of virulence factors and the common features of the diseases caused by these species in their respective hosts. The bordetellae are usually defined as obligate, non-invasive parasites of the respiratory tracts of warm-blooded animals, including birds, with a predilection for the respiratory ciliated epithelium. This definition has been challenged by a number of recent observations. For example, the ability of Bordetella spp. to regulate virulence factor expression in response to external signals strongly suggests that they have alternative habitats where such regulation would be an advantage. These habitats may be intracellular, since it has been shown that B. pertussis, B. parapertussis and B. bronchiseptica can invade and survive within host cells, or they may be in other sites within the same or different hosts. Recent DNA fingerprinting studies of B. pertussis have revealed hitherto unsuspected heterogeneity amongst isolates which could be reflected in antigenic differences between strains. Some of these new perspectives on Bordetella pathogenicity may have implications for pertussis vaccine development.     

Early production of a chemotactic factor to T lymphocytes by peritoneal macrophages

Supernatant fluids (SNF) were obtained from peritoneal exudate adherent cells stimulated in vitro with sheep red blood cells (SRBC) or BCG, and SNF collected at 6 and 24 hr were able to induce the migratory responses of rat leukocytes from the spleen and peripheral blood. The production of these SNF was dependent on protein active synthesis upon in vitro antigenic stimulation. The chemotactic activity from 6-hr SNF was inhibited by using several proteolytic enzymes and temperatures. We found the macrophages to be the producer cell of this activity, while the T cells were the target cells. The chemotactic activity from 6-hr SNF was found not to be due to IL-1. Six-hour chemotactic activity has not been reported previously.     

Human T-lymphocyte chemotactic activity: nature and production in response to antigen

Previous studies have shown that supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells and isolated Leu-2 suppressor/cytotoxic T cells are chemotactic for Leu-3 helper/inducer T cells. The current study shows that lymphocyte chemotactic factor (LCF) is also produced following antigen (tetanus toxoid) challenge of mononuclear cells obtained from recently immunized human donors. LCF was detected in 24-hr supernatants from mononuclear cells challenged with tetanus and was produced maximally at 48 hr. Tetanus toxoid challenge of mononuclear cells obtained from individuals whom had not received a tetanus immunization for 7 to 10 years prior to testing showed little or no production of LCF. Serial studies of these individuals following a tetanus booster immunization showed that LCF was produced by antigen-challenged mononuclear cells obtained 1-5 days postimmunization, was produced optimally 5-15 days postimmunization, and was still produced by antigen-challenged mononuclear cells obtained 6 weeks later. Fractionation of mononuclear cells from immunized donors into glass wool nonadherent lymphocytes, T lymphocytes, and non-T lymphocytes showed that tetanus-toxoid-induced LCF was produced by nonadherent lymphocytes and T cells but not non-T cells. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T-cell subpopulations showed that LCF production by antigen-challenged isolated subpopulations was limited to the Leu-2 suppressor/cytotoxic T-cell subset. Characterization of both Con A and tetanus toxoid-induced LCF by gel filtration on Sephadex G-100 demonstrated the presence of two peaks of LCF corresponding to molecular weights of approximately 14,000-17,000 and 40,000-50,000.     

Opsonizing activity of sera from animals immunized with pertussis vaccine

Opsonizing activity of guinea pig blood serum containing mercaptoethanol-resistant pertussis antibodies was studied in vitro on a model of microorganism ingestion by the mononuclears of the guinea pig peritoneal exudate. There were revealed distinct differences in the serum activity depending on the phagocytosis object. The blood serum of hyperimmunized rabbits stimulated the ingestion of Bordetella pertussis by mononuclears of guinea pigs--normal and immunized with pertussis vaccine. The blood sera of hyperimmunized guinea pigs and of mice immunized with pertussis vaccine twice displayed opsonins to B. pertussis. The blood sera of animals immunized with pertussis vaccine inhibited the staphylococcus ingestion by the peritoneal exudate mononuclears of guinea pigs, both normal and those immunized with pertussis vaccine.     

Antigens of Bordetella pertussis. IV. Effect of heat, merthiolate, and formaldehyde on histamine-sensitizing factor and protective activity of soluble extracts from Bordetella pertussi.s

Munoz, J. (Rocky Mountain Laboratory, Hamilton, Mont.), and B. M. Hestekin. Antigens of Bordetella pertussis. IV. Effect of heat, Merthiolate, and formaldehyde on histamine-sensitizing factor and protective activity of soluble extracts from Bordetella pertussis. J. Bacteriol. 91:2175-2179. 1966.-Both histamine-sensitizing and protective activities of soluble preparations from Bordetella pertussis cells are destroyed by heating at 80 C for 0.5 hr. The histamine-sensitizing activity appeared to be more susceptible to inactivation by heat than the protective activity. Formaldehyde in a final concentration of 0.5% rapidly diminished the histamine-sensitizing ability of saline extract (SE) held at 37 C. The protective activity was clearly more resistant to inactivation by formaldehyde at similar temperature. The inactivating action of formaldehyde was slower when the concentration of SE was increased or when the mixture was kept at 2 to 5 C. Merthiolate in a final concentration of 1:10,000 had no demonstrable deleterious effects on either protective or histamine-sensitizing activity of SE.     

Attenuated Bordetella pertussis vaccine candidate BPZE1 promotes human dendritic cell CCL21-induced migration and drives a Th1/Th17 response

New vaccines against pertussis are needed to evoke full protection and long-lasting immunological memory starting from the first administration in neonates--the major target of the life-threatening pertussis infection. A novel live attenuated Bordetella pertussis vaccine strain, BPZE1, has been developed by eliminating or detoxifying three important B. pertussis virulence factors: pertussis toxin, dermonecrotic toxin, and tracheal cytotoxin. We used a human preclinical ex vivo model based on monocyte-derived dendritic cells (MDDCs) to evaluate BPZE1 immunogenicity. We studied the effects of BPZE1 on MDDC functions, focusing on the impact of Bordetella-primed dendritic cells in the regulation of Th and suppressor T cells (Ts). BPZE1 is able to activate human MDDCs and to promote the production of a broad spectrum of proinflammatory and regulatory cytokines. Moreover, conversely to its parental wild-type counterpart BPSM, BPZE1-primed MDDCs very efficiently migrate in vitro in response to the lymphatic chemokine CCL21, due to the inactivation of pertussis toxin enzymatic activity. BPZE1-primed MDDCs drove a mixed Th1/Th17 polarization and also induced functional Ts. Experiments performed in a Transwell system showed that cell contact rather than the production of soluble factors was required for suppression activity. Overall, our findings support the potential of BPZE1 as a novel live attenuated pertussis vaccine, as BPZE1-challenged dendritic cells might migrate from the site of infection to the lymph nodes, prime Th cells, mount an adaptive immune response, and orchestrate Th1/Th17 and Ts responses.