Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O₂ ^•-) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O₂ ^•- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O₂ ^•- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O₂ ^•- than those from controls. Nifedipine treatment considerably decreased O₂ ^•- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O₂ ^•- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O₂ ^•- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.     

Chimeric HIV-1 Envelope Glycoproteins with Potent Intrinsic Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Activity*

HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env_GM-CSF chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env_GM-CSF proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env_GM-CSF should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.     

Granulocyte-macrophage colony-stimulating factor increases the proportion of circulating dendritic cells after autologous but not after allogeneic hematopoietic stem cell transplantation

Background aimsGranulocyte–macrophage (GM) colony-stimulating factor (CSF) has been used as an adjuvant in cancer immunotherapy. We tested the hypothesis that GM-CSF (Leukine^®; sargramostim) improves immune reconstitution after hematopoietic stem cell transplantation (HSCT) based on our prior in vitro work that demonstrated the pro-inflammatory effects of GM-CSF on dendritic cells (DC).MethodsGM-CSF was administered to donors, along with standard granulocyte (G) CSF, during stem cell mobilization, and to recipients from the day prior to transplant until engraftment. Eighteen patients consented to the GM-CSF⁺ protocol and were compared with 17 matched controls undergoing HSCT during the same time period (GM-CSF^?).ResultsNumbers of white blood cells (WBC) and CD34⁺ stem cells in the graft were comparable to controls. Surprisingly, contrary to our hypothesis, the allogeneic donor graft had significantly decreased numbers of CD3⁺ T cells and their subsets (CD4⁺, CD4⁺ CD45RA⁺, CD4⁺ CD45RO⁺, CD8⁺ and CD8⁺ CD45RO⁺), DC (both myeloid and plasmacytoid) and natural killer (NK) cells (CD16⁺ CD56⁺). In the GM-CSF arm, following allogeneic transplantation, the levels of DC, T cells and NK cells did not increase with treatment. Conversely, autologous transplant patients receiving GM-CSF had a higher proportion of DC at the time of engraftment.ConclusionsThese findings demonstrate that administration of GM-CSF improves DC reconstitution after autologous rather than allogeneic HSCT.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on itsin vitro andin vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Mast cell growth factor (C-Kit Ligand) in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3:in vivo hemopoietic effects in irradiated mice compared toin vitro effects

In the presence of hemopoietic cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), mast cell growth factor (MGF; also known as steel factor, stem cell factor, and c-kit ligand) has proven to be a potent hemopoietic regulatorin vitro. In these studies, we examined thein vivo effects of MGF in combination with GM-CSF or GM-CSF plus IL-3. Effects were based on the ability of these cytokines to stimulate recovery from radiation-induced hemopoietic aplasia. Female B6D2F1 mice were exposed to a sublethal 7.75-Gy dose of⁶⁰Co radiation followed by subcutaneous administration of either saline, recombinant murine (rm) MGF (100g/kg/day), rmGM-CSF (100g/kg/day), rmIL-3 (100g/kg/day), or combinations of these cytokines on days 1–17 postirradiation. Recoveries of bone marrow and splenic spleen colony-forming units (CFU-s), granulocyte macrophage colony-forming cells (GM-CFC), and peripheral white blood cells (WBC), red blood cells (RBC) and platelets (PLT) were determined on days 14 and 17 during the postirradiation recovery period. MGF administered in combination with GM-CSF or in combination with GM-CSF plus IL-3 either produced no greater response than GM-CSF alone or down-regulated the GM-CSF-induced recovery. These results sharply contrasted results ofin vitro studies evaluating the effects of these cytokines on induction of GM-CFC colony formation from bone marrow cells obtained from normal or irradiated B6D2F1 mice, in which MGF synergized with GM-CSF or GM-CSF plus IL-3 to increase both GM-CFC colony numbers and colony size. These studies demonstrate a dichotomy between MGF-induced effectsin vivo andin vitro and emphasize that caution should be taken in attempting to predict cytokine interactionsin vivo in hemopoietically injured animals based onin vitro cytokine effects.Abbreviations GM-CSF Granulocyte-Macrophage Colonly-Stimulating Factor - IL-3 Interleukin-3 - MGF Mast Cell Growth Factor - SCF Stem Cell Factor - rm Recombinant Murine - CFU-s Colony Forming Unit-Spleen - GM-CFC Granulocyte Macrophage Colony-Forming Cell - WBC White Blood Cells - RBC Red Blood Cells - PLT Platelets - SLF Steel Factor - G-CSF Granulocyte Colonly-Stimulating Factor - IL-1 Interleukin-1 - IL-6 Interleukin-6 - Epo Erythropoietin - CFC Colony-Forming Cell - Sl Steel - BFU-e Erythroid Burst Forming Units - s.c Subcutaneous - PEG Polyethyleneglycol - PIXY321 GM-CSF/IL-3 Fusion Protein     

Monocytes from Chronic HBV Patients React In Vitro to HBsAg and TLR by Producing Cytokines Irrespective of Stage of Disease

Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogenous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the in vitro effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected in vivo. We observed that in vitro HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier in vivo exposure to HBsAg does not affect the in vitro response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production in vitro. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive in vivo monocyte activation.     

Role of T cell subsets in the modulation of Mycobacterium avium growth within human monocytes

Mycobacterium avium frequently causes disseminated disease in patients with advanced AIDS with low CD4 counts. The effects of T lymphocyte on intracellular M. avium replication were examined. Plastic adherent monocytes and nonadherent lymphocytes were separated from peripheral blood mononuclear cells. After infection with M. avium, monocytes were cultured with or without autologous lymphocytes (1-10 cells/monocyte) for up to 7 days. Addition of lymphocytes to M. avium-infected monocytes significantly decreased intracellular M. avium growth after 7 days culture (n = 11, P < 0.01, paired t test) and increased IFN-gamma production compared to monocytes alone. Neutralizing IFN-gamma partially abrogated lymphocyte activity. CD4 depletion diminished anti-mycobactericidal effects and CD8(+) lymphocytes increased intracellular M. avium growth (P < 0.05, n = 5, t test). These data suggest that interactions between monocytes and nonadherent cell fractions such as CD4(+) T cells and NK cells are important in intracellular M. avium growth modulation in monocytes from healthy humans.     

Coronary artery bypass grafting is associated with immunoparalysis of monocytes and dendritic cells

Coronary artery bypass grafting (CABG) triggers a systemic inflammatory response that may contribute to adverse outcomes. Dendritic cells (DC) and monocytes are immunoregulatory cells potentially affected by CABG, contributing to an altered immune state. This study investigated changes in DC and monocyte responses in CABG patients at 5 time‐points: admission, peri‐operative, ICU, day 3 and day 5. Whole blood from 49 CABG patients was used in an ex vivo whole blood culture model to prospectively assess DC and monocyte responses. Lipopolysaccharide (LPS) was added in parallel to model responses to an infectious complication. Co‐stimulatory and adhesion molecule expression and intracellular mediator production was measured by flow cytometry. CABG modulated monocyte and DC responses. In addition, DC and monocytes were immunoparalysed, evidenced by failure of co‐stimulatory and adhesion molecules (eg HLA‐DR), and intracellular mediators (eg IL‐6) to respond to LPS stimulation. DC and monocyte modulation was associated with prolonged ICU length of stay and post‐operative atrial fibrillation. DC and monocyte cytokine production did not recover by day 5 post‐surgery. This study provides evidence that CABG modulates DC and monocyte responses. Using an ex vivo model to assess immune competency of CABG patients may help identify biomarkers to predict adverse outcomes.     

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.     

Mesenchymal stromal cells impair the differentiation of CD14(++) CD16(-) CD64(+) classical monocytes into CD14(++) CD16(+) CD64(++) activate monocytes

Background aimsMesenchymal stromal cells (MSC) possess immunomodulatory activity both in vitro and in vivo. However, little information is available regarding their function during the initiation of immunologic responses through their interactions with monocytes. While many studies have shown that MSC impair the differentiation of monocytes into dendritic cells and macrophages, there are few articles showing the interaction between MSC and monocytes and none of them has addressed the question of monocyte subset modulationMethodsTo understand better the mechanism behind the benefit of MSC infusion for graft-versus-host treatment through monocyte involvement, we performed mixed leucocyte reactions (MLR) in the presence and absence of MSC. After 3 and 7 days, cultures were analyzed by flow cytometry using different approachesResultsMSC induced changes in monocyte phenotype in an MLR. This alteration was accompanied by an increase in monocyte counting and CD14 expression. MSC induced monocyte alterations even without contact, although the parameters above were more pronounced with cell–cell contact. Moreover, the presence of MSC impaired major histocompatibility complex (MHC) I and II, CD11c and CCR5 expression and induced CD14 and CD64 expression on monocytes. These alterations were accompanied by a decrease in interleukin (IL)-1β and IL-6 production by these monocytes, but no change was observed taking into account the phagocytosis capacity of these monocytesConclusionsOur results suggest that MSC impair the differentiation of CD14⁺⁺ CD16^? CD64⁺ classical monocytes into CD14⁺⁺ CD16⁺ CD64⁺⁺ activated monocytes, having an even earlier role than the differentiation of monocytes into dendritic cells and macrophages.     

Defective monocyte to macrophage maturation in human immunodeficiency virus infection

To look for possible defects in cells of the monocyte/macrophage system, blood monocytes from patients infected with human immunodeficiency virus (HIV) were cultured on hydrophobic Teflon for 7 days and their ability to differentiate into mature macrophages in the presence of serum was followed. The following parameters were studied as indicative of successful terminal maturation: (1) the expression of maturation-associated antigens (transferrin receptor, surface transferrin, the BA-2 antigen, MAX antigens), (2) the disappearance of the MOP15 antigen, and (3) a more than 20-fold increase in intracellular ferritin concentration. It was found that the patients′ blood monocytes did not differentiate in vitro but rather remained immature precursor cells. If the same holds true in vivo, the results could indicate that the pathophysiology of the acquired immunodeficiency syndrome (AIDS) may be, to a large extent, linked with the functional consequences of this impaired monocyte-to-macrophage maturation.     

Anti-allergic effects of His-Ala-Gln tripeptide in vitro and in vivo

We examined the inhibitory effects of HAQ (His-Ala-Gln) peptide on type-1 allergy in vitro and in vivo. HAQ peptide inhibited β-hexosaminidase release and intracellular Ca²⁺ levels of rat basophilic leukemia RBL-2H3 cells. Oral administration of a HAQ peptide-added diet (1 mg/mouse/administration) to C3H/HeJ mice for 14 days led to significant suppression of allergic symptoms, but did not reduce allergen-specific IgE or IgG₁.     

Human Monocyte Recognition of Adenosine-Based Cyclic Dinucleotides Unveils the A2a Gαs Protein-Coupled Receptor Tonic Inhibition of Mitochondrially Induced Cell Death

Cyclic dinucleotides are important messengers for bacteria and protozoa and are well-characterized immunity alarmins for infected mammalian cells through intracellular binding to STING receptors. We sought to investigate their unknown extracellular effects by adding cyclic dinucleotides to the culture medium of freshly isolated human blood cells in vitro. Here we report that adenosine-containing cyclic dinucleotides induce the selective apoptosis of monocytes through a novel apoptotic pathway. We demonstrate that these compounds are inverse agonist ligands of A2a, a G_αs-coupled adenosine receptor selectively expressed by monocytes. Inhibition of monocyte A2a by these ligands induces apoptosis through a mechanism independent of that of the STING receptors. The blockade of basal (adenosine-free) signaling from A2a inhibits protein kinase A (PKA) activity, thereby recruiting cytosolic p53, which opens the mitochondrial permeability transition pore and impairs mitochondrial respiration, resulting in apoptosis. A2a antagonists and inverse agonist ligands induce apoptosis of human monocytes, while A2a agonists are antiapoptotic. In vivo, we used a mock developing human hematopoietic system through NSG mice transplanted with human CD34⁺ cells. Treatment with cyclic di-AMP selectively depleted A2a-expressing monocytes and their precursors via apoptosis. Thus, monocyte recognition of cyclic dinucleotides unravels a novel proapoptotic pathway: the A2a G_αs protein-coupled receptor (GPCR)-driven tonic inhibitory signaling of mitochondrion-induced cell death.     

Stimulation of Locomotion of Peripheral Blood Monocytes by Human Plasma Fibronectin

The motility of human peripheral blood granulocytes and monocytes in response to human plasma fibronectin was quantified by an in vitro assay using blind-well chemotaxis chambers. Purified fibronectin under nondenaturing conditions produced increased migration of granulocytes only at concentrations higher than 100 nm, and induced increased chemotactic and random locomotion of monocytes at concentrations higher than 0.1 nm. The monocyte migration-inducing activity of fibronectin was concentration dependent, and was strongly inhibited by low concentrations of colchicine (100 nm–100 μm). These findings suggest the possibility that plasma fibronectin serves as a chemotactic stimulus for monocytes in vivo and attracts these cells to sites of microscopic tissue injury where plasma fibronectin is deposited.     

Biosynthesis in isolatedAcetabularia chloroplasts II. Plastid pigments

Summary The plastid pigments — chlorophylls and carotenoids — of the alga,Acetabularia, have been chromatographically separated and identified. These pigments were found to become radio-active during incubations of an isolated chloroplast fraction with¹⁴CO₂. Specific activity calculations indicate that appreciable amounts of synthesis were occurringin vitro. The phytol and porphyrin moieties of chlorophyll a were both radioactive; thus the pigments were being formed completely from recent photosynthetic products. A comparison of the incorporation of¹⁴CO₂ into plastid pigmentsin vivo andin vitro suggests that the isolated chloroplasts form the pigments at their normalin vivo rates.     

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium,a Pro-Atherogenic Mechanism

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.     

Immunomodulatory effects ofBacillus firmus

3-day-old miniature piglets were stimulatedin vivo withBacillus firmus by the intraperitoneal or intragastric route for 1 d. Cells containing IgA and IgG₂ were detected in the ileum in all stimulated but not in control animals. The frequency of blood CD3⁺ cells increased after intraperitoneal administration ofB. firmus, the ratio of polymorphonuclears to lymphocytes increased in all stimulated piglets.B. firmus induced antitumor immunity in rats with transplanted Yoshida sarcoma cells. Granular lymphocytes and dead tumor cells were found in peritoneal exudate of stimulated animals.B. firmus induced IFN-γ synthesis in human blood lymphocytes stimulatedin vitro for 1 d. The amount of TNF-α produced by these stimulated human peripheral blood mononuclears (PBMC) was lower than that of PBMC stimulated with some other bacterial immunomodulators. Cells containing TGF-β or IL-8 were not found in human PBMC stimulated withB. firmus.     

Intracellular Concentrations of Potassium,Chloride, and Protons during Differentiation of Tobacco Male Gametophyte

Changes in intracellular pH, K⁺and Cl^–concentrations were measured in pollen grains, anther loculi, and whole anthers during in vivodifferentiation of male gametophyte of Nicotiana tabacumL. The effects of extracellular K⁺and Cl^–on intracellular pH regulation were also studied during development of pollen grains in vitro. Ion-selective electrodes and X-ray microanalysis were used to measure ion concentrations, and microfluorometry was used to measure pH. The intracellular pH and [K⁺] decreased at the mid- and late binucleate stages of pollen grain development, while [Cl^–] increased. At the same stages, K⁺and Cl^–concentrations in locular liquid were found to increase. The intracellular pH in pollen grains, isolated at the mid-binucleate stage, decreased during in vitrodevelopment in the medium containing 50 mM KCl. It was suggested that changes in ionic composition of the external medium can regulate the intracellular pH during development of pollen grain in vivo.