Sendai virus stimulates chemiluminescence in mouse spleen cells.

Sendai virus stimulates chemiluminescence within a few seconds after it is added to a suspension of mouse spleen cells. Virus rendered non infectious by irradiation with ultraviolet light induces a similar burst of chemiluminescence. Heating or pronase treatment of the virus abrogate this reaction, as does sonication of the cells before the addition of the virus. The ability of the virus to stimulate chemiluminescence is correlated with its hemagglutination, neuraminidase, cell fusion and hemolytic properties. It is suggested that Sendai virus-induced chemiluminescence is initiated by the interaction of the virus envelope spike glycoproteins with the cell membrane.     

Quantification of hemokinin-1 peptide production and secretion from mouse B cells

Hemokinin-1 is a recent addition to the family of mammalian tachykinins and is thought to play an important role in B cell and T cell lymphopoiesis. The mRNA coding for this peptide was expressed in some B lymphocyte cell lines including 70Z/3.12, ABE-8.1/2, and RAW8.1 cells, suggesting the possibility that hemokinin-1 may function in an autocrine or paracrine manner in these cells. Therefore, we quantified secretion of this peptide from the 70Z/3.12 cell line expressing hemokinin-1 mRNA. Despite a sensitive radioimmunoassay, we were surprised to find that hemokinin-1 secretion from confluent cells was below the level of detection of this assay. Furthermore, cell lysates routinely demonstrated a low or undetectable immunoreactive peptide. Collectively these studies show a limited production of hemokinin-1 peptide by transformed B cells.     

The role of macrophages in B cell tolerance. I. Antigen-specific failure of Thy-1, Ly-2 negative adherent cells from tolerant mice to reconstitute immunocompetence in adherent cell deficient spleen cells

T-Cell-independent B-cell tolerance to the hapten derivatives of carboxymethyl cellulose (CMC) or methyl cellulose (MC) appears to be controlled by Thy-1-, Ly-2- adherent (A) cells contained in the spleen or peritoneal fluid. Immunocompetence in nonadherent (NA) normal spleen cells could be restored in vitro by irradiated A cells from normal mice. However, NA cells reconstituted with irradiated A cells derived from hapten specifically tolerant mice failed to respond to the same hapten, but responded normally to an immunogenic challenge with another unrelated antigen. A cells that had been preincubated at 4 degrees C with hapten derivatized MC also failed to restore immunocompetence. While preincubation of unfractionated spleen cells with the tolerogen under the same conditions resulted in B-cell unresponsiveness, such treatment of NA cells failed to render B cells tolerant. Treatment of A cells from tolerant mice with the reducing agent potassium iodide (KI) in vitro restored their capacity to render cultures of NA cells immunocompetent to the relevant hapten. Moreover, treatment with KI of spleen cells from mice injected with the tolerogen was shown to render them responsive. We suggest that B-cell tolerance induced by hapten derivatives of CMC and MC is mediated by suppressive macrophages contained among A cells. Certain subpopulations of macrophages are known to exert cytotoxic effects upon target cells by the release at close range of oxidating agents. We postulate that hapten derivatized CMC and MC, through unique properties of the carrier, bind to and possibly activate macrophages rendering them specifically suppressive for hapten binding B cells.     

Crystallization of human beta-hexosaminidase B.

beta-Hexosaminidase is a lysosomal hydrolase that is important in the metabolism of sphingoglycolipids. beta-Hexosaminidase B and beta-hexosaminidase A are the major isozymes in normal human tissue. beta-Hexosaminidase B is a homodimer of beta subunits, and beta-hexosaminidase A is a heterodimer composed of an alpha and a beta subunit. Crystals of beta-hexosaminidase B (M(r) 112,000) have been grown using the handling drop technique. They are elongated hexagonal prisms with maximum dimensions of 0.2 mm x 0.2 mm x 0.7 mm. The space group is P6(1)22 (or enantiomorph); the unit cell dimensions are a = b = 114.2 A, c = 402.2 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one dimer per asymmetric unit. Crystals diffract to 3.2 A resolution.     

Characterization of erythropoietin-like activity from mouse spleen cells

Supernatants from mouse spleen cell cultures contain a factor which acts in a similar manner to erythropoietin (Ep) to stimulate the formation of 2-day erythroid (CFU-E) colonies in vitro from bone marrow or fetal liver cells. Analysis of conditioned media by high performance liquid chromatography (HPLC) on anion exchange, reverse phase, molecular size exclusion, and hydroxyapatite columns demonstrated that the erythropoietin-like activity (EpLA) has different biochemical characteristics to mouse Ep from anemic mouse serum. In addition, EpLA has a molecular weight (Mr), of 20,000 daltons determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), compared to 42,000 for mouse Ep. Partially purified EpLA was found to be active in vivo as well as in vitro. Highly purified preparations of gamma-interferon, Multilineage hemopoietic growth factor (Multi HGF), Interleukin-2 (IL-2), IL-1, and colony stimulating factor 1 (CSF-1) did not support CFU-E colony formation. Thus, it was established that EpLA could not be attributed to other known components of spleen cell conditioned medium. Titration of mouse Ep and EpLA suggests that only a portion of the Ep-responsive CFU-E population in fetal liver is sensitive to EpLA.     

Helodermin stimulates glucagon secretion in the mouse

Helodermin is structurally similar to VIP (vasoactive intestinal peptide) and PHI (peptide histidine isoleucine). Since VIP and PHI both stimulate insulin and glucagon secretion, we investigated the effects of helodermin on insulin and glucagon secretion in the mouse, both in the basal state and during administration of glucose and the cholinergic agonist carbachol. After intravenous injection at dose levels between 0.5 and 8.0 nmol/kg, helodermin markedly enhanced basal plasma glucagon levels, for example at 8 nmol/kg from 139 +/- 14 to 421 +/- 86 pg/ml (p less than 0.001) after 6 minutes, without affecting basal plasma insulin levels. Together with glucose (2.8 mmol/kg), helodermin (2 and 8 nmol/kg) augmented plasma glucagon levels but had no effect on plasma insulin levels. When injected together with the cholinergic agonist carbachol (0.16 mumol/kg), helodermin markedly potentiated the increase in plasma glucagon levels (more than three-fold; p less than 0.001), again without affecting the plasma insulin levels. Combined alpha- and beta-adrenoceptor blockade (yohimbine + L-propranolol) reduced the augmenting effect of helodermin on glucagon secretion by approximately 60%. It is concluded helodermin stimulates glucagon secretion in the mouse by an effect that is partially antagonized by combined alpha- and beta-adrenoceptor antagonism.     

Effect of hydrocortisone and bacterial lipopolysaccharide on colony-stimulating activity production from mouse marrow adherent cells, spleen cells and peritoneal macrophages in vitro

The effect of hydrocortisone (HC) on colony-stimulating activity (CSA) production from mouse bone marrow adherent cells, spleen cells and peritoneal macrophages with or without bacterial lipopolysaccharide (LPS) stimulation was studied. CSA in the supernatant from bone marrow adherent cells incubated with HC was found to be five times higher than CSA from cultures without LPS stimulation. In contrast, the CSA production by spleen cells and peritoneal macrophages were significantly suppressed by HC in both LPS-stimulated and non-stimulated cultures. These studies suggest that the effect of HC on CSA production was quite different depending on the target cells.     

Phorbol ester stimulates amylase secretion from rat parotid cells

Phorbol myristate acetate (PMA), a potent activator of Ca2+- and phospholipid-dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose-response studies, PMA stimulated amylase release independently of db-cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic-cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.     

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100ng/ml) for 18h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1μM) or INSL3 antagonist (bovine and human; 100ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.     

DCEBIO stimulates Cl- secretion in the mouse jejunum

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl^– secretory response of the mouse jejunum using the Ussing short-circuit current (I_sc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in I_sc (EC₅₀ 41 ± 1 µM). Pretreating tissues with 0.25 µM forskolin reduced the concentration-dependent increase in I_sc by DCEBIO and increased the EC₅₀ (53 ± 5 µM). Bumetanide blocked (82 ± 5%) the DCEBIO-stimulated I_sc consistent with Cl^– secretion. DCEBIO was a more potent stimulator of Cl^– secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated I_sc by >80% indicating the participation of CFTR in the DCEBIO-stimulated I_sc response. Clotrimazole reduced DCEBIO-stimulated I_sc by 67 ± 15%, suggesting the participation of the intermediate conductance Ca²⁺-activated K⁺ channel (IK_Ca) in the DCEBIO-activated I_sc response. In the presence of maximum forskolin (10 µM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in I_sc of 43 ± 5 µA/cm² and then falling to a steady-state response of 17 ± 10 µA/cm² compared with DCEBIO control tissues (61 ± 6 µA/cm²). The forskolin-stimulated I_sc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated I_sc, providing evidence that DCEBIO increased Cl^– secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl^– secretion of the mouse jejunum and that DCEBIO targets components of the Cl^– secretory mechanism. 1-ethyl-2-benzimidazolinone; forskolin; glibenclamide; clotrimazole; H89     

Interleukin-6 stimulates gonadotropin-releasing hormone secretion from rat hypothalamic cells

There are reports that both interleukin-1 beta and interleukin-6 (IL-6) stimulate the release of adrenocorticotropin through stimulation of hypothalamic corticotropin-releasing factor. We established a primary culture system for hypothalamic neurons producing gonadotropin-releasing hormone (GnRH) and examined whether IL-6 stimulated their GnRH secretion. We demonstrated immunohistochemically that some of these neurons contained GnRH-like immunoreactivity. In primary cultures of these GnRH neurons, we found that the calcium ionophore A23187 stimulated GnRH secretion in a dose- and time-dependent manner. These hypothalamic cells secreted IL-6 spontaneously, producing about 10 ng/l in 24 h, and their IL-6 secretion was significantly stimulated by E2 at 10(-9)-10(-8) mol/l. This stimulatory effect was observed within 3 h. IL-6 also stimulated the release of GnRH in a dose- and time-dependent manner, and these effects of IL-6 were significantly blocked by anti-IL-6 antiserum. These results suggest that the central action of IL-6 on the GnRH neurons may be an important physiological event in the hypothalamus.     

Separation of various B-cell subpopulations from mouse spleen. I. Depletion of B cells by rosetting with glutaraldehyde-fixed, anti-immunoglobulin-coupled red blood cells.

Mouse spleen cells were depleted of immunoglobulin (Ig)-bearing B cells by rosetting with glutaraldehyde-fixed, tannic acid-treated RBC coupled with antibody to mouse Ig (anti-Ig) and removing the rosetted cells by density gradient centrifugation. The method was routinely greater than 90% effective in removing B cells as assayed by the failure of anti-Ig rosette-depleted primed spleen cells to generate antibody-producing cells in vitro in response to specific antigen or of anti-Ig rosette-depleted nonprimed spleen cells to generate a polyclonal antibody response. T cells were not removed by the rosetting procedure as measured by helper T-cell activity. The greater effectiveness of the rosetting procedure in removing potential IgG-secreting, non-IgM-bearing B cells is shown relative to other commonly used B-cell depletion procedures. Because the RBC in the rosetting reagent are fixed with glutaraldehyde, the rosetting reagent is stable for many months. Such stability makes constantly available a convenient means for B-cell removal, as well as reducing consumption of antisera.     

Oleate stimulates secretion of apolipoprotein B-containing lipoproteins from Hep G2 cells by inhibiting early intracellular degradation of apolipoprotein B.

Studies were conducted to explore the effects of oleate addition on the secretion of apolipoprotein B (apoB)-containing lipoproteins from Hep G2 cells. Whether oleate was added simultaneously with [3H]-leucine or added to prelabeled cells, the rate of secretion of apoB was stimulated more than 100% within 40 min. When oleate was withdrawn from the cells, the rate of secretion returned to the prestimulated rate within 40 min. These observations suggested that oleate affects apoB secretion early in the secretory pathway. When the effects of oleate on apoB secretion were studied in pulse-chase experiments, it was observed that although apoB synthesis was not affected, apoB intracellular degradation was significant inhibited by oleate. In the absence of oleate, 58% of apoB synthesized during the labeling period was degraded within 20 min, before secretion of apoB into the media had begun, whereas only 29% of labeled apoB was degraded intracellularly during this same time period when oleate was present. Thus, it appears that oleate rapidly stimulates the secretion of apoB by protecting nascent apoB from degradation early in the secretory pathway. Furthermore, stimulation of apoB secretion was observed over a range that includes physiological concentrations of oleate, from 0.1 mM (oleate: bovine serum albumin ratio = 0.45) to 0.8 mM (oleate: ratio = 3.6), suggesting that exogenous oleate could be a physiological modulator of apoB secretion.     

Differential inhibition of beta-hexosaminidase A and beta-hexosaminidase B by suramin

The trypanocidal drug suramin is a potent inhibitor of β-hexosaminidase A with a K_i of about 4.5 μM, and to a lesser extent of β-hexosaminidase B (K_i 31.5 μM). β-Hexosaminidase B remained active in the presence of 1.0 mM suramin whereas the activity of β-hexosaminidase A was completely inhibited at 0.1 mM.     

Separation of various B-cell subpopulations from mouse spleen. II. Depletion of antigen-specific B cells by rosetting with glutaraldehyde-fixed, antigen-coupled red blood cells.

Mouse spleen cells were depleted of antigen-specific B cells (ASC) by rosetting with glutaraldehyde-fixed RBC coupled with specific antigen. Removal of the rosetted cells by density gradient centrifugation resulted in the depletion of more than 99% of all the ASC. Depletion was shown by the failure of rosette-depleted, primed spleen cells to generate antigen-specific antibody-producing cells. Functional assays showed that neither macrophages or helper T cells were removed. In comparison to other procedures used to deplete of ASC, rosetting with antigen-coupled RBC has advantages in terms of recovery, simplicity of the procedure, and efficiency of ASC depletion.     

Synthesis and secretion of immunoglobulin G by lymphocytes from cultured mouse spleen cells is not affected by heat shock

Short-term hyperthermic episodes (in vivo and in vitro) alter gene expression in mammalian lymphocytes, resulting in the enhanced synthesis of a select group of polypeptides--the heat-shock proteins--and the depressed synthesis of many normally synthesized polypeptides. Such alterations could have profound implications to an individual if the appropriate functioning of lymphocytes within the immune response was compromised by a depression in immunoglobulin synthesis during naturally occurring periods of hyperthermia, such as fever. In the present study we asked if heat-shock affects the facultative synthesis and secretion of immunoglobulin G by cultured mouse lymphocytes. We found that the quantity of immunoglobulin G synthesized and secreted by these cells is not affected by heat-shock treatments sufficient to induce the synthesis of heat-shock proteins.     

The differential ability of splenic adherent cells from B6.lpr mice to induce suppressor T cells

Hapten-coupled splenic adherent cells or resident peritoneal cells from autoimmune B6.lpr mice that are over 5 mo of age fail to induce first-order inducer suppressor T cells (Ts1). However, the same population of hapten-coupled cells can induce both delayed-type hypersensitivity responses and third-order effector suppressor T cells (Ts3). Thus, splenic and peritoneal antigen-presenting cells from B6.lpr mice display a defined defect in the ability to induce certain suppressor T cell responses. The cellular defect in Ts1 induction is controlled by the lpr gene, since age-matched congenic B6 mice do not display this defect. The splenic adherent cell defect is temporarily correlated with the autoimmunity that develops in B6.lpr animals. The antigen-presenting defect in the B6.lpr splenic adherent population for Ts1 induction is reversible by culturing the cells in interferon-gamma. The results are discussed as an illustration of the relationship between experimental models of autoimmunity and defects in a suppressor T cell cascade.     

IFN-gamma stimulates IgG2a secretion by murine B cells stimulated with bacterial lipopolysaccharide

rIFN-gamma strikingly enhances the secretion of IgG2a by murine splenic B cells stimulated with bacterial LPS in vitro and concomitantly suppresses the production of IgG3, IgG1, IgG2b, and IgE while sparing IgM secretion. IFN-gamma stimulates highly purified B cell populations to secrete IgG2a, strongly suggesting that it acts directly on B cells. It increases the frequency of precursors of IgG2a-expressing soft agar colonies and enhances the number of IgG2a+ cells in colonies indicating that it both increases the frequency of precursors of IgG2a+ cells and enhances the number of IgG2a+ daughter cells emerging from each precursor. IFN-gamma completes its action within the first 24 to 48 h of a 6-day culture with LPS and its addition cannot be delayed beyond the first 48 h. Preincubation of resting B cells in the presence of IFN-gamma leads to a time dependent increase, up to 42 h, in IgG2a secretion upon subsequent addition of LPS. IFN-gamma can exert this action on resting B cells that have been selected for absence of membrane IgG expression by cell sorting. The promotion of IgG2a secretion appears to be a specific property of IFN-gamma in that IFN-alpha, IFN-beta, IL-1, IL-2, IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, granulocyte-CSF, and CSF-1 fail to enhance IgG2a secretion by LPS-stimulated B cells.