Pertussis toxin induces lymphocytosis in rhesus macaques

Abstract: Lymph nodes and other solid tissues of the immune system are the principal sites for antigen presentation and lymphocyte activation. Lymphocytes in peripheral blood recognize the high endothelial venules within lymphoid tissues and cross from blood to tissue by the process of extravasation. Pertussis toxin is known to block extravasation and cause lymphocytosis in murine models but has not been studied extensively in nonhuman primates. We used intravenous injection of soluble pertussis toxin to induce a transient lymphocytosis in rhesus monkeys. The increase in total white blood cells was proportionally greater for lymphocytes than for polymorphonuclear cells and the CD4+ lymphocyte subpopulation increased more than the CD8+ cell population. The presence of immature polymorphonuclear cells suggested some activation of bone marrow. Clinical chemistry studies revealed an effect of pertussis toxin on liver function. Pertussis toxin is a powerful immunomodulatory agent that can disrupt and reorganize solid lymphoid tissues.     

Differences in prostaglandin formation between thymocyte subpopulations.

The concentration of prostaglandin E and the activity of prostaglandin synthetase were determined in mature and immature mouse thymocytes. Hydrocortisone resistant thymocytes, or thymocytes separated from the immature cell population after agglutination of the latter by peanut agglutinin served as a source of mature thymocytes. It was found that mature thymocytes contain a much higher concentration of prostaglandin E and have an increased activity of prostaglandin synthetase than immature cells.     

Studies on thymocyte subpopulations in guinea pigs. X. Rosette-forming ability of thymocyte subpopulations before and after incubation in vitro

Thymocytes spontaneously proliferating in vitro were labelled with 3H-thymidine, and the distribution of label among rosette-forming cells (RFC+) and nonrosetting cells (RFC-), as well as in populations differing in buoyant density, was measured by liquid scintillation counting and autoradiography before and after incubation for 24 h. Initially most labelled cells (88%) belonged to the low-density (1a) subpopulation, the majority being RFC+. After incubation for 24 h, low-density nonrosetting thymocytes (1a, RFC-) contained the highest amount of label. A decreased rosette formation occurred not only in labelled cells but also in the population as a whole, and in separately incubated high-density cells. The decreased rosette formation was mainly caused by a change in rosette-forming ability of viable high-density cells, however in part also by decreased viability. A shift from low to high density occurred among labelled cells during incubation and was shown to occur in both RFC+ and RFC-. The decreased rosette formation of labelled cells during in vitro culture contrasts with the increase earlier observed in vivo and may therefore represent affinity alterations or a down-regulation of the rosette receptor in vitro. We conclude that the observed changes in density, but not in rosette-forming ability, may reflect normal differentiation.     

3-O-methylglucose transport by rat thymocyte subpopulations.

Rat thymocytes can be separated into two subpopulations by centrifugation for 20 minutes at 1,600 g in an 18/26/36% (w/v) discontinuous gradient of bovine serum albumin. Approximately 13% of the cells band at the 18/26% interface (light cells) while the remaining cells band at the 26/36% interface (heavy cells). In vitro and in vivo studies of 3H-thymidine incorporation indicate that the light cells are 2- to 3-fold enriched in the rapidly dividing lymphoblast subpopulation of thymocytes as compared to heavy cells. Light cells transport the non-metabolizable glucose analogue 3-O-methylglucose (3-MeGlc) approximately four times faster than heavy cells. The time course of 3-MeGlc uptake is biphasic for light, heavy and unfractionated thymocytes. While the half-times of the rapid (1 minute) and slow (20-45 minute) phases of uptake are similar for all three types of cells, the contributions of the rapid phase to total uptake are 50% for light cells, 20% for unfractionated thymocytes and 10% for heavy cells. The results show that 3-MeGlc transport activity differs markedly within certain thymocyte subpopulations. The correlation between the contributions of the rapid phase of uptake and the proportion of lymphoblasts in the thymocyte fractions suggests that the lymphoblast and small lymphocyte subpopulations might be responsible for the rapid and slow phase of 3-MeGlc uptake, respectively.     

Differences in prostaglandin formation between thymocyte subpopulations

The concentration of prostaglandin E and the activity of prostaglandin synthetase were determined in mature and immature mouse thymocytes. Hydrocorthisone resistant thymocytes, or thymocytes separated from the immature cell population after agglutination of the latter by peanut agglutinin served as a source of mature thymocytes. It was found that mature thymocytes contain a much higher concentration of prostaglandin E and have an increased activity of prostaglandin synthetase than immature cells.     

Development of persistent lymphocytosis in cattle is closely associated with DRB2

Correspondence to: H. A. Lewin.     

Pertussis toxin-induced cytokine differentiation and clonal expansion of T cells is mediated predominantly via costimulation

Pertussis toxin (PTX) has potent immunologic adjuvant activity in vivo and concomitantly enhances both T helper type (Th1) and Th2 cytokine responses. The PTX-induced enhancement of Th1 and Th2 immunity is mediated via the activation of antigen presenting cells (APCs), but the underlying mechanism is not known. Here we asked whether the adjuvant activity of PTX on T cell immunity was mediated by cytokines and/or costimulatory signals. The results show that in vivo blockade of CD28-CD80/86 costimulation essentially abrogated PTX-mediated enhancement of antigen-specific Th1 and Th2 responses. Blockade of CD40L-CD40 interactions was less efficient in inhibiting PTX-mediated enhancement of Th1 and Th2 responses. In contrast, the adjuvant activity of PTX was not mediated via cytokines, because neither Th1 nor Th2 responses were substantially impaired in mice deficient for IL-12, IFN-gamma, IL-4, IL-5, or IL-6. Collectively, the data suggest that PTX mediates its adjuvant effects on T cell cytokine differentiation and clonal expansion via the modulation of costimulatory molecules on APCs. Understanding the costimulatory pathways targeted by PTX could lead to the design of novel adjuvants that selectively induce Th1 or Th2 immunity.     

Characterization of murine thymocyte subpopulations reacting with Dolichos bifloris agglutinin

Reactivity of murine thymocytes with the Dolichos bifloris agglutinin (DBA) was examined using flow cytometry. This agglutinin, which has nominal specificity for alpha-linked N-acetyl-D-galactosamine residues, labels 1-4% of unfractionated BALB/c thymocytes. Among thymocyte subpopulations identified on the basis of CD4/CD8 labeling, DBA bound to a substantial percentage of the CD4-8- thymocyte subpopulation. In addition, small populations of thymocytes expressing CD4 and/or CD8 were also labeled with DBA, but with less intensity then seen on many CD4-8- cells. Within the CD4-8- thymocyte population, labeling with DBA was positively correlated with reactivity with J11d antibody, the expression of IL-2 receptor and high levels of the homing receptor molecule, MEL-14. Reactivity with DBA was negatively correlated with the expression of Lyt-1, V beta 8 TCR determinants, and CD3.     

Pertussis toxin treatment in vivo is associated with a decline in G-protein beta-subunits

The effects of pertussis toxin on the steady-state levels of G-protein alpha- and beta-subunits were investigated both in vitro and in vivo. The steady-state level Go alpha, a major substrate for pertussis toxin-catalyzed ADP-ribosylation, was unaltered by pertussis toxin treatment for periods up to 100 h for 3T3-L1 cells in culture or up to 3 days in vivo. In 3T3-L1 cells pertussis toxin treatment did not alter levels of Gs alpha-subunits; in S49 cells the level of Gs alpha-subunits declined moderately following by pertussis toxin treatment. The steady-state levels of G beta-subunits, in contrast, were found to decline to less than 50% of the normal cellular complement following pertussis toxin treatment in vitro and in vivo. Inhibitory control of adenylate cyclase, pertussis toxin-catalyzed ADP-ribosylation of Gi alpha and Go alpha, and the GTP-dependent shift in agonist-specific binding to beta-adrenergic receptors were attenuated or abolished within 5 h of pertussis toxin treatment, representing "early" effects of the toxin. Stimulatory regulation of adenylate cyclase, in contrast, displayed a progressive enhancement that was first observed 4 h after pertussis toxin treatment, increasing thereafter up until 100 h, the last time point measured. This progressive enhancement of the stimulatory pathway of adenylate cyclase was not manifest at the level of stimulatory receptors, since the Kd and Bmax for one such receptor, the beta-adrenergic receptor, were shown to be unaltered in toxin-treated cells. Furthermore, the potentiation of stimulation of adenylate cyclase was observed in cells stimulated by the beta-adrenergic agonist isoproterenol and PGE1 alike. The progressive enhancement of the stimulatory pathway correlated best with the decline in G beta-subunit levels that occurs following pertussis intoxication. The changes in both of these parameters occur "late" (12-48 h), as compared to the early events that occur within 5 h. Pertussis toxin action appears to be composed of two, temporally distinct, groups of effects. Pertussis toxin-catalyzed ADP-ribosylation of G alpha-subunits, attenuation of the inhibitory regulation of adenylate cyclase, and attenuation of the ability of GTP to induce an agonist-specific shift in receptor affinity are members of the early group of effects. The second group of late effects includes the decline in G beta-subunit levels and the progressive enhancement of the stimulatory pathway of adenylate cyclase. This enhanced stimulatory control at these later times cannot be explained by the attenuation of the inhibitory pathway occurring early, but rather appears as G beta-subunit levels decline.     

Cytotoxic and fluorescent assays for thymocyte subpopulations differing in surface thy-1 level.

A number of analytical techniques for distinguishing and separating the "high theta" "low theta" subpopulations of mouse thymocytes have been compared. A differential cytotoxic assay was compared to a quantitative immunofluorescent assay on individual cells using flow cytofluorometry and cell sorting. Conventional anti-Thy-1 antisera were compared with a monoclonal IgM anti-Thy-1. The monoclonal reagent greatly improved both types of assay, eliminated a number of artifacts and allowed either procedure to be used to give a clear distinction, based on Thy-1 level, between the two subpopulations. The distribution of Thy-1 on thymocytes is bimodal, rather than continuous. These separate "high theta" and "low theta" categories each includes a population a population of dividing cells.     

Chromosomal terminal methylation status is associated with gut microbiotic alterations

Molecular and Cellular Biochemistry - We explored the association of fecal bacterial species and somatic telomere changes in patients with chronic disease. The results showed that the length of the...     

Studies on thymocyte subpopulations in guinea pigs. VIII. Characterization of a thymocyte population resistant to the cytotoxic effect of normal rabbit serum

Guinea pig thymocytes were incubated with normal rabbit serum, which resulted in the death of a great majority of the cells. The remaining rabbit serum-resistant cells, representing less than 10% of the thymocytes, contained euchromatic DNA and were of intermediate size and low density. Functional tests indicated that they were enriched in immunologically mature cells, which responded to the mitogenic lectins phytohemagglutinin and concanavalin A, and were depleted of immature, spontaneously proliferating cells and in cells responding to the thymocyte growth peptide. The described procedure for enrichment of immunologically mature thymus cells in guinea pigs may become useful since glucocorticoid treatment, used in mice for enrichment of mature thymocytes, cannot be used for this purpose in guinea pigs.     

Ultrastructural alterations in glomerulopathy associated with hydatidosis in sheep.

In the present study we investigated the ultrastructural alterations occurring in the renal glomeruli of sheep with hydatidosis. Renal samples from 39 sheep, 34 with hydatidosis and 5 without parasitosis, were examined by transmission electron microscopy. Additionally, biochemical analysis was performed by determining serum concentrations of creatinine, urea, total protein and albumin. The ultrastructural alterations identified were the presence of dense mesangial, subendothelial and intra-membranous deposits, mesangial cell proliferation with areas showing segmental sclerosis and interposition of mesangial cells with the formation of a neomembrane. Biochemical analysis revealed a significant increase in total serum protein in the experimental group compared with the control. Our results demonstrated that glomerulonephritis associated with hydatidosis in sheep can be classified into four categories: minimal lesions, mesangial glomerulonephritis, segmental and focal glomerulonephritis and membranoproliferative glomerulonephritis, being membranoproliferative and mesangial glomerulonephritis the most predominant categories.     

Biosynthesis and maturation of ribosomal RNA in thymocyte subpopulations of X-irradiated rats

A study was made of RNA biosynthesis and maturation in the control and irradiated thymocyte fractions isolated in a ficoll-paque gradient. The post-irradiation impairment of rRNA processing was manifested by the enhancement of pre-rRNA biosynthesis and the increase in 18S rRNA "wastage" during the first hours following X-irradiation. The changes were most pronounced in the thymocyte fraction sedimenting in a gradient zone with the density of above 1.077.     

Allergic rhinitis is associated with complex alterations in high-density lipoprotein composition and function

Despite strong evidence that high-density lipoproteins (HDLs) modulate the immune response, the role of HDL in allergies is still poorly understood. Many patients with allergic rhinitis (AR) develop a late-phase response, characterized by infiltration of monocytes and eosinophils into the nasal submucosa. Functional impairment of HDL in AR-patients may insufficiently suppress inflammation and cell infiltration, but the effect of AR on the composition and function of HDL is not understood. We used apolipoprotein (apo) B-depleted serum as well as isolated HDL from AR-patients (n = 43) and non-allergic healthy controls (n = 20) for detailed compositional and functional characterization of HDL. Both AR-HDL and apoB-depleted serum of AR-patients showed decreased anti-oxidative capacity and impaired ability to suppress monocyte nuclear factor-κB expression and pro-inflammatory cytokine secretion, such as interleukin (IL)-4, IL-6, IL-8, tumor necrosis factor alpha and IL-1 beta. Sera of AR-patients showed decreased paraoxonase and cholesteryl-ester transfer protein activities, increased lipoprotein-associated phospholipase A2 activity, while lecithin-cholesterol acyltransferase activity and cholesterol efflux capacity were not altered. Surprisingly, apoB-depleted serum and HDL from AR-patients showed an increased ability to suppress eosinophil effector responses upon eotaxin-2/CCL24 stimulation. Mass spectrometry and biochemical analyses showed reduced levels of apoA-I and phosphatidylcholine, but increased levels of apoA-II, triglycerides and lyso-phosphatidylcholine in AR-HDL. The changes in AR-HDL composition were associated with altered functional properties. In conclusion, AR alters HDL composition linked to decreased anti-oxidative and anti-inflammatory properties but improves the ability of HDL to suppress eosinophil effector responses.     

Studies on thymocyte subpopulations in guinea pigs. III. Physical and functional characterization of six subpopulations separated by density gradient centrifugation and PNA binding

Thymocytes were separated according to increasing buoyant density into the three subpopulations Ia (25% of recovered cells), Ib (20%) and II (55%), and according to binding to peanut agglutinin (PNA)into PNA+ (65%) and PNA- cells (35%). The frequency of PNA+ was 56% in Ia, 60% in Ib and 66% in population II. Electronic cell volume determinations disclosed mean volumes of 160 fl for Ia, 130 fl for Ib and 100 fl for population II. PNA+ and PNA- cells were very similar as regards cell volume. Thus, PNA+ and PNA- cells are remarkably uniformly distributed among cell categories of different density and cell volume. The rapidly cycling thymocytes, regarded as the most immature cells in the thymus, and the target cells for a thymocyte growth factor both belonged to the PNA+ cells of population Ia. The mitogen-responsive thymocytes also belonged to population Ia, but were PNA-. The largest subpopulation of thymocytes, apparently corresponding to the small, non-cycling cortical cells, were recovered as PNA+ cells of population II.     

Early endotoxin tolerance is associated with alterations in bone marrow-derived macrophage precursor pools

Early endotoxin tolerance has been defined as the transient period after an initial sublethal exposure to LPS during which a normally responsive individual is rendered hyporesponsive. Little is known about the cellular mechanisms that underlie this phenomenon. In this study, an early tolerance system was established by the injection of mice with 25 micrograms of E. coli K235 LPS. Maximal hyporesponsiveness in response to a challenge injection was observed 3 to 4 days after the initial injection, and normal responsiveness returned by 8 days after the initial exposure to LPS. Further experiments described herein demonstrate that the acquisition and maintenance of the tolerant state coincides temporally with an increase in the number of macrophage progenitor cells in the bone marrow. Cell-sizing profiles of the bone marrow cells from tolerized mice indicate an enrichment for a population of cells that are significantly larger than in bone marrow preparations from control mice. By density gradient sedimentation, it was shown that the denser population of cells from tolerized mice contained the increased numbers of progenitor cells, which, by cytology, were immature monocytic cell types. The increased numbers of macrophage progenitors was sustained after a second (challenge) injection of LPS. These results indicate that early endotoxin tolerance is associated with an increase in a population of bone marrow cells that is enriched for macrophage progenitors and suggests the possibility that the lack of responsiveness observed during the hyporesponsive period is related to a failure of these immature cell types to respond to LPS.