Effect of the microtubule stabilising agent taxol on leishmanial protozoan parasites in vitro

Taxol, a mitotic spindle toxin, was found to selectively inhibit the proliferation of Leishmania donovani in vitro at nanomolar concentrations with an IC50 of 35 nM. Concentrations of taxol as high as 50 nM, however, did not affect J774A.1 murine macrophages. Taxol (30 nM) also inhibited amastigote multiplication within a J774A.1 macrophage cell line when used in a 10-day experiment. It resulted in the in vitro assembly of L. donovani microtubules in a dose-dependent manner. When promastigotes were exposed to different concentrations of taxol for 24 h, cells were largely blocked in the G2-M phase of the cell cycle and there was a marked reduction in the percentage of cells in the S phase. The selective nature of taxol action against the parasite and its effectiveness in controlling amastigote multiplication emphasise its use as a promising chemotherapeutic against kala-azar.     

Possible role of the 34-kilodalton hyaluronic acid-binding protein in visceral Leishmaniasis.

Leishmania donovani, the causative organism of human visceral leishmaniasis, invades host macrophages through its interaction with the cell surface molecules of target cells. The presence of a cell surface protein (Mr 34 kDa) having specific affinity toward hyaluronan (HA), a major extracellular matrix component, has been previously reported in macrophage cell lines. In order to identify the possible role of this HA-binding protein (HABP) in leishmaniasis, initially we demonstrated its overexpression in spleen, liver, macrophages, and serum of hamsters infected with L. donovani. We further observed higher levels of HABP in the macrophage cell line J774.G8 upon infection with L. donovani. Finally, we observed a significant increase in the level of HABP in the serum of patients with kala-azar. In order to understand its functional role in leishmaniasis, we report here a significant inhibition of cellular phosphorylation of HABP in hamster macrophages infected with L. donovani. Interestingly, the 34-kDa HABP was shown to bind with 2 proteins of promastigotes as well as amastigotes of L. donovani (with molecular masses of 55 kDa and 30 kDa respectively), suggesting a possible role for HABP in adhesion during the interaction of promastigotes and macrophages.     

Requirement of A1-a for bacillus Calmette-Guérin-mediated protection of macrophages against nitric oxide-induced apoptosis

The role of apoptosis in regulating the course of intracellular microbial infection is not well understood. We studied the relationship between apoptotic regulation and bacillus Calmette-Guérin (BCG) treatment in murine peritoneal exudate macrophages (PEM) and the J774 macrophage cell line. In both PEM and J774 cells, mRNA expression of the anti-apoptotic gene, A1, was selectively induced by BCG treatment as compared with other bcl2 family members (bcl-w, bcl-2, bcl-xl, bcl-xs, bax, bak, bad). In PEM, A1 expression was maximal by 8 h postinfection and was abrogated by the proteasomal inhibitor MG-132. The induction was independent of protein synthesis as well as the p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways and did not require live organism. Three genes encoding closely related isoforms of A1 were all expressed; however, the A1-a isoform displayed the greatest fold induction in PEM. BCG-induced A1 expression was associated with protection of host macrophages from NO-mediated apoptosis in both PEM and J774 cells. BCG-mediated protection was abrogated in PEM derived from A1-a(-/-) mice, indicating a requirement of A1-a for survival of inflammatory macrophages.     

Mouse resident peritoneal macrophages partially control in vitro infection with Coxiella burnetii phase II

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is a small, dimorphic, obligate intracellular bacterium, sheltered within large, acidified, and hydrolase-rich phagosomes. Although several primary and established cell lines, macrophage-like cells, and primary macrophages from other species have been infected with C. burnetii, the infection of mouse primary macrophages has not been sufficiently characterized. In this report quantification of DAPI (4', 6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy, and transmission electron microscopy were used to compare the infection of three mouse-derived cells, L929 fibroblasts, J774 macrophage-like cells, and resident peritoneal macrophages, with a phase II clone of C. burnetii known to be non-virulent for mammals. Infected peritoneal phagocytes differed from L929 or J774 cells in that: (a) large vacuoles took longer to appear (3-5 d instead of 2), and were only found in a subset (20-30%) of macrophages, as opposed to in more than 70% of the other cells; (b) total and vacuole-associated relative bacterial loads in L929 and J774 cells were several-fold higher than in peritoneal macrophages; (c) estimated doubling times of the bacteria were about 68 h in the primary macrophages, 18 h in J774 and 22 h in L929 cells. Thus, mouse resident peritoneal macrophages control both the formation of the large vacuoles and the intracellular proliferation of C. burnetii phase II.     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.     

An unusual pro-inflammatory role of interleukin-10 induced by arabinosylated lipoarabinomannan in murine peritoneal macrophages

Various species of Mycobacteria produce a major cell wall-associated lipoglycan, called Lipoarabinomannan (LAM), which is involved in the virulence of Mycobacterial species. In this study, we tried to establish the role of the increased IL-10 secretion under Arabinosylated-LAM (Ara-LAM) treatment, the LAM that induces apoptosis in host macrophages or PBMC. We have studied the survival and apoptotic factors by western blotting, and estimated nitrite generation by Griess reaction, quantified iNOS mRNA by semi-quantitative RT-PCR, and ultimately the fate of the cells were studied by Flow Cytometric Analysis of AnnexinV-FITC binding. As per our observations, neutralization of released IL-10 in C57BL/6 peritoneal macrophages prior to Ara-LAM treatment, as well as macrophages from IL-10 knockout (KO) mice treated with Ara-LAM, showed significant down regulation of pro-apoptotic factors and up regulation of survival factors. These effects were strikingly similar to those when peritoneal macrophages were subjected to TNF-α and IL-12 neutralization followed by Ara-LAM-treatment. However, under similar conditions virulent Mannosylated-LAM (from Mycobacterium tuberculosis) treatment of macrophages clearly depicts the importance of IL-10 in the maintenance of pathogenesis, proving its usual immunosuppressive role. Thus, from our detailed investigations we point out an unusual pro-inflammatory action of IL-10 in Ara-LAM treated macrophages, where it behaves in a similar manner as the known Th1 cytokines TNF- α and IL-12. This work is financed by the Council of Scientific and Industrial Research (CSIR), Govt. of India.     

Isolation of the 66-kilodalton polypeptide from promastigotes of Leishmania donovani as a ligand molecule for binding to macrophages.

A 66-kDa major plasma membrane-associated molecule of promastigotes of Leishmania donovani (UR6) was purified by affinity chromatography. The immunoreactivity of the 66-kDa molecule was lost upon exposure to heat or treatment with trypsin. The metaperiodate oxidation significantly reduced its immunoreactivity. The 66-kDa molecule is, therefore, glycoprotein in nature. With a fluorescent probe, the 66-kDa molecule was found to be located on the tip of flagellum and on the kinetoplast. The exposure of promastigotes of L. donovani to monospecific anti-66-kDa antibodies significantly reduced the percentage of macrophages with attached promastigotes in the cultured cell line (J774G8). The data suggested that promastigotes of L. donovani utilize the 66-kDa molecule in recognizing and as ligand for binding to macrophages.     

Leishmania donovani flagellum-specific epitopes mediating host-parasite interactions

Abstract Monoclonal antibodies were developed against flagellar components of promastigotes of Leishmania donovani . The monoclonal antibody produced by clone A₁₁ (mAb A₁₁) recognised epitopes in the polypeptides with molecular weights of 86, 66 and weakly 53 kDa. These epitopes were found to be distributed along the flagellum and at the anterior end of promastigotes. The mAb A₁₁ of IgG₁ isotype strongly agglutinated the promastigotes of L. donovani . The prior treatment of promastigotes of L. donovani with mAb A₁₁ resulted in a significant ( P < 0.001) reduction in the attachment of promastigotes to cultured mouse peritoneal macrophages of line J774G8. The affinity-purified epitopes identified by mAb A₁₁ were recognised by human sera of cases of visceral leishmaniasis. The present study suggest that flagellar-specific epitopes mediate host-parasite interactions and, therefore, the role of these epitopes in the disease process is speculated.     

Killing of Leishmania donovani by activated liver macrophages from resistant and susceptible strains of mice

Leishmania donovani is an obligate intracellular protozoan parasite of macrophages; liver macrophages are, however, the only population of cells which express the resistant Lsh gene phenotype when these cells are infected in vitro. It was of interest to study in vitro the action of Con A-stimulated spleen cell lymphokines (LK) to protect or to cure liver macrophages from infection by L. donovani. Liver and peritoneal macrophages (PEC) from resistant (C57L/J) and susceptible (C57BL/6J) mice were infected in vitro with promastigotes before or after LK treatment; the percentage of infected macrophages was determined 4, 24, 48 and 72 h post-infection. Both macrophage populations were protected or cured by treatment with lymphokines; the cells of the resistant strain were protected or cured more effectively than those of the susceptible strain. The capacity for cure or for protection following LK treatment of liver and PEC macrophages was similar within each strain. Supernatants from the IL-2-produced MLA-144 cell line had no effect to protect or cure macrophages. This study indicates that the response of macrophages to the action of LK is also important in determining the susceptibility of mice to L. donovani; this model in vitro provides a good approximation of the response of macrophages to therapy.     

Production of prostaglandin D2 by murine macrophage cell lines

Several tumor-derived murine macrophage cell lines were evaluated in vitro as cloned prototypes of tissue macrophages for their ability to metabolize arachidonic acid. Unexpectedly, two cell lines, J774A.1 and WR19M.1, rapidly converted exogenous 14C-arachidonic acid (AA) to a single major prostaglandin metabolite. The compound, PGD2, was positively identified by TLC, HPLC, and GC-MS. The enzymatic formation of the PGD2 was shown by inhibition of its formation by indomethacin and reduced formation of 14C-PGD2 from 14C-PGH2 in boiled cells. When J774A.1 cells were prelabeled with 3H-AA, cultured for 24 hours, and stimulated with lipopolysaccharide (LPS), PGD2 was again the predominant product. No other tumor derived cell lines, including several other murine macrophage lines, produced significant amounts of PGD2. Elicited and activated murine peritoneal macrophages produced only small amounts of PGD2, but resident peritoneal macrophages produced modest amounts of PGD2. Exaggerated formation of PGD2 by J774A.1 and WR19M.1 cells may be a consequence of neoplastic transformation or the clonal expansion of a minor subpopulation of normal tissue macrophages.     

In vitro and in vivo induction of nitric oxide by murine macrophages stimulated with Bordetella pertussis

Abstract Nitric oxide (NO) exhibits potent antimicrobial activity in vitro. The function of NO in host defenses in vivo, however, is presently unclear. Experiments were undertaken to determine the production of NO in vitro from murine peritoneal and alveolar macrophages, and murine macrophage cell line (J774A.1) stimulated with Bordetella pertussis or pertussis toxin (PT). In addition, we determined circulating levels of NO in the sera and bronchoalveolar lavage (BAL) fluids of mice infected intranasally with B. pertussis . The results of this study showed that in vitro murine peritoneal macrophages induce production of NO in response to B. pertussis and PT. In addition, murine macrophage cell line, J774A.1 also induces NO production after stimulation with B. pertussis . NO production was also detected in alveolar macrophages from mice infected intranasally with B. pertussis . Finally, a significant increment of circulating levels of NO was noted, in the sera but not in the BAL fluids, of mice infected intranasally with B. pertussis .     

Cholesterol esterification as a limiting factor in accumulation of cell cholesterol: a comparison of two J774 macrophage cell lines

It was previously reported by Tabas et al. that J774 macrophages, unlike mouse peritoneal macrophages, accumulate large amounts of cholesteryl esters when incubated with native low-density lipoprotein (LDL). Comparison of the cell line (designated J774A.2) used in those experiments with its parent line (J774A.1) indicates that it is a variant with a greater rate of cholesterol esterification. This large difference in cholesterol esterification was accompanied by only a small difference in rates of LDL uptake and degradation by the J774A.2 line. The J774A.2 cells have become a variant line through either mutation or selection which has enhanced its susceptibility to foam cell formation by its markedly increased ability to esterify cholesterol.     

Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F-actin accumulated progressively around phagosomes containing wild-type L. donovani promastigotes during the first hour of phagocytosis. Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F-actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.     

Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 macrophages

J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH4Cl induced a 12-fold increase in TGase secretion. NH4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.     

The interaction of Naegleria fowleri amoebae with murine macrophage cell lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

A study to evaluate the effect of nootropic drug-piracetam on DNA damage in leukocytes and macrophages

Piracetam is a nootropic drug that protects neurons in neuropathological and age-related diseases and the activation and modulation of peripheral blood cells in patients with neuropathological conditions is well known. Therefore, in the present study, in vivo, ex vivo, and in vitro tests were conducted to investigate the effect of piracetam on leukocytes and macrophages. Lipopolysaccharide (LPS) causes oxidative DNA damage; thus, in the present study, LPS was used as a tool to induce DNA damage. In vivo experiments were conducted on Sprague Dawley rats, and piracetam (600mg/kg, oral) was provided for five consecutive days. On the fifth day, a single injection of LPS (10mg/kg, i.p.) was administered. Three hours after LPS injection, blood leukocytes and peritoneal macrophages were collected and processed, and a variety of different assays were conducted. Ex vivo treatments were performed on isolated rat blood leukocytes, and in vitro experiments were conducted on rat macrophage cell line J774A.1. Cell viability and the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage were estimated in untreated (control) and piracetam-, LPS- and LPS+piracetam-treated leukocytes and macrophages. In vivo experiments revealed that rats pretreated with piracetam were significantly protected against LPS-induced increases in ROS levels and DNA damage. Ex vivo isolated leukocytes and J774A.1 cells treated with LPS exhibited augmented ROS levels and DNA damage, which were attenuated with piracetam treatment. Thus, the present study revealed the salutary effect of piracetam against LPS-induced oxidative stress and DNA damage in leukocytes and macrophages.     

Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with unopsonized prey

Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin. Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation. Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey. In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey. We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown. We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.     

Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate

Cholesterol esterification by acyl CoA:cholesterol acyl transferase (ACAT) in macrophages is a key process in atheroma foam cell formation. However, the process of cholesterol substrate delivery to ACAT is not well defined. In this study, J774 macrophages, which form foam cells with native low density lipoprotein (LDL), were labeled with [3H]cholesterol-containing liposomes. Most (80-90%) of the cholesterol label could be converted by cholesterol oxidase to cholestenone, suggesting plasma membrane localization; only 0.6% of the label was in cholesteryl ester (CE). In cells chased for 6 h in medium lacking LDL, the distribution of label was essentially unchanged, whereas in cells chased with LDL, 28% of the label was incorporated into CE concomitant with a decrease in cholestenone label to 50%. [3H]Cholesterol-labeled mouse peritoneal macrophages incubated with acetyl-LDL, and both J774 and mouse peritoneal macrophages incubated with 25-hydroxy-cholesterol, also showed a shift of label from cholestenone to CE. Similar results were found when cellular cholesterol was biosynthetically labeled with [3H]mevalonate. The percentage of cholesterol substrate for ACAT in LDL-treated J774 macrophages which originates from endogenous cellular pools (versus that originating from LDL itself) is approximately 50%. We conclude that upon activation of ACAT in macrophages, there is a novel process whereby a cholesterol oxidase-accessible pool of cellular cholesterol, presumably plasma membrane cholesterol, is translocated to ACAT in the endoplasmic reticulum.