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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 2  |  Page : 151-156

Effect of levamisole on alkaline phosphatase activity and immunoglobulin secretion of lipopolysaccharide-stimulated murine splenic lymphocytes


1 Department of Biotechnology, Government College Autonomous, Rajamahendravaram, Andhra Pradesh, India
2 Department of Biochemistry, Mahatma Gandhi University, Nalgonda, Telangana, India

Date of Web Publication23-Nov-2017

Correspondence Address:
Nageshwari Badgu
Department of Biotechnology, Government College Autonomous, Rajamahendravaram, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_83_17

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  Abstract 


Background: Alkaline phosphatases (APase) are a group of enzymes whose activity increases above normal levels in conditions of diseases such as cancer. In the present study, the role of APase using mitogen-stimulated murine lymphocytes was investigated. Methods: U266B1 cells and RPMI 8226 cultures were kept at 37°C in a humidified incubator with 5% CO2. The cultures were pulsed with 0.5 μCi of 3H-thymidine and were harvested onto glass fiber filter using Skatron automatic cell harvester. The dried filters were transferred into toluene-based scintillation cocktail, and the radioactivity was measured using Beckman scintillation counter. APase activity was determined by p-nitrophenol phosphate hydrolysis. The intracellular immunoglobulin E (IgE) content was quantified by western blot assay. U266 B1 and RPMI 8226 were cultured at 0.25 × 106/ml with and without 1 mM levamisole for 48 h, and 15 ml of the culture supernatant was collected and lyophilized. Electrophoresis was carried out on 30 μl of the dialyzed samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and the gels were silver stained. Results: APase may be involved in the constitutive proliferation as well as in Ig secretion of myeloma cells. It was observed that murine splenic lymphocytes showed an increase in proliferative response concomitant with an increase in the APase activity and Ig secretion upon mitogenic stimulation (0.5–2.5 mM, P > 0.05). Conclusion: Levamisole significantly inhibited the APase activity when added to the lipopolysaccharide-stimulated cells at the initiation of the culture. Significance of the present study is discussed in the light of existing literature.

Keywords: Immunoglobulin secretion, levamisole, mitogenic stimulation, myeloma cells


How to cite this article:
Badgu N, Merugu R. Effect of levamisole on alkaline phosphatase activity and immunoglobulin secretion of lipopolysaccharide-stimulated murine splenic lymphocytes. Biomed Biotechnol Res J 2017;1:151-6

How to cite this URL:
Badgu N, Merugu R. Effect of levamisole on alkaline phosphatase activity and immunoglobulin secretion of lipopolysaccharide-stimulated murine splenic lymphocytes. Biomed Biotechnol Res J [serial online] 2017 [cited 2019 Oct 20];1:151-6. Available from: http://www.bmbtrj.org/text.asp?2017/1/2/151/219114




  Introduction Top


The B lymphocyte is the principal cell involved in mediating specific humoral immune response to infection. Before the onset of immune response, lymphocytes are quiescent, nondividing cells circulating in the blood and reside in the secondary lymphoid organs. On stimulation with an antigen, B lymphocytes go through three distinct phases of cell cycle – early activation (G1 phase), proliferation (S phase), and differentiation to immunoglobulin (Ig)-secreting plasma cells.[1]

Alkaline phosphatase (APase) is a membrane-bound glycoprotein which has been shown to be expressed in B lymphocytes upon activation and has been used as a marker of B-cell activation.[2],[3],[4],[5] It has also been shown that the enhancement of APase activity correlates with proliferation and differentiation in murine B-cells.[3],[6] Further studies have shown that APase activity is expressed in B-lymphocytes committed to proliferation and the activity increases further in the antibody-secreting cells.[7] Although APase activity has been studied for many years in different tissues, its physiological role has remained largely enigmatic and is still under investigation. Earlier, it has been hypothesized that APase may be involved in the transport of Ig molecules and in phosphorylation/dephosphorylation reactions.[8],[9]

It has been proposed that APase may have a physiological role in the activated B-cell in terms of:

  1. As a phosphotyrosine phosphatase in the early stages of signaling
  2. Involvement in the proliferation and differentiation
  3. Transport of Ig molecules from the plasma cells.


We used cell lines, i.e. U266B1 cells and RPMI 8226 to study the role of B lymphocyte APase in proliferation and differentiation and to assess if the APase activity can be targeted to inhibit the proliferation of these cells. Immune response and disease resistance of Clarias fuscus aquaculture upon exposure to levamisole was studied.[10] An uncompetitive inhibitor of APase, levamisole, has been chosen for the study as in addition to having the property of inhibiting APase, it has also been found to have anticancer properties. Levamisole is known to increase leukocyte count and Ig levels in boars.[11] Treatment with levamisole and colchicine can result in a significant reduction of interleukin-6 (IL-6), IL-8, or tumor necrosis factor (TNF)-alpha level in patients with mucocutaneous type of Behcet's disease.[12] The hepatoprotective effect of stem cells and levamisole against carbon tetrachloride-induced liver fibrosis was also reported.[13] In the present experiments, levamisole has been used to study the role of APase in proliferation, differentiation leading to Ig secretion.

The objectives are to study the effect of levamisole on proliferative response and APase activity of lipopolysaccharide (LPS)-stimulated murine splenic lymphocytes, on Ig secretion by LPS-stimulated murine splenic lymphocytes, and on Ig secretion by myeloma cell lines.


  Methods Top


Preparation of mouse splenic lymphocytes

Procedure

The mouse was killed using ether anesthesia. Its left side of the abdomen was then wiped with 70% alcohol, a cut was made through the skin, and the spleen was then separated from the vessels and connecting tissue with scissors. The spleen was then placed in RPMI 1640 medium on a stainless steel mesh kept on a  Petri dish More Details and then minced into small pieces. The minced tissue was teased carefully using teasers with regular addition of medium onto the tissue. The cell suspension was transferred to a sterile tube for the large clumps to settle down. The cell suspension was taken into a fresh tube and centrifuged at 400 g for 10 min after which it was kept on ice. The pellet was then resuspended in RPMI 1640 and was carefully layered on Histopaque (d = 1.077, half the volume of the suspension) and centrifuged at 800 g for 10 min. The lymphocytes separated were at the interface of RPMI 1640, gradient solution was collected carefully, and the centrifugation step was repeated twice as described above. The final cell pellet was suspended in RPMI 1640 with 5% fetal calf serum (FCS).

Proliferative response

Splenic lymphocytes, 0.2 × 106 cells/well, were cultured with 5 μg LPS and with varying concentrations of levamisole. The cultures were pulsed with 0.5 μCi of 3H-thymidine for the last 24 h of 72-h culture period and were processed as described.

3H-thymidine incorporation assay

Cells were cultured in triplicate in 200 μl of complete medium in 96-well flat-bottomed microtiter plates. Cultures were kept at 37°C in a humidified incubator with 5% CO2. The cultures were pulsed with 0.5 μCi of3 H-thymidine for the past 24 h of 72-h culture period and were harvested onto glass fiber filter using Skatron automatic cell harvester. The dried filters were transferred into toluene-based scintillation cocktail, and the radioactivity was measured using Beckman scintillation counter. APase activity was determined by p-nitrophenol (p-NP) hydrolysis. The absorbance of p-NP produced was measured at 405 nm. Cells were dispensed into wells of microtiter plate and centrifuged at 450 g for 10 min at 4°C. The cells were suspended in 0.9% saline, and the centrifugation step was repeated. To the pellet, 180 μl of 1 mg/ml p-NP phosphate in 0.1M bicarbonate buffer with 2 mM MgCl2 was added. Incubation was carried out at 37°C in a humidified incubator. After 30 min, the reaction was stopped by the addition of 20 μl of 1N NaOH and the absorbance was measured at 405 nm using ELISA plate reader. The amount of p-NP released was calculated from a standard graph. The results were expressed as nmoles of p-NP released/0.2 × 106 cells.

  1. Culture supernatant of LPS-stimulated murine splenic lymphocytes was collected at the end of 7 days, and the IgM was detected by dot blot assay
  2. U266B1 cells were seeded at a concentration of 5 × 104 cells/well in 200 μl medium in a 96-well microtiter plate. Culture supernatant of untreated and levamisole-treated U266 B1 cells was collected at the end of 48 h, and the IgE was detected by dot blot assay.


Procedure

Cell-free supernatant containing Ig 5ul sample was spotted onto the nitrocellulose membrane and dried for 1 h at room temperature. The membrane was then washed thrice with tris buffered saline (TBS) for 5 min each time. The membrane was then blocked with 5% peptide solution made in TBS tween (TBST) for 1 h. It was then incubated for 4–6 h with either anti-human IgM- Horseradish Peroxidase (HRPO) conjugate or anti-human IgE–APase conjugate antibody to Ig, diluted 1:2000 in 1% w/v of peptide solution (w/v) at room temperature. Then, the blot was washed with TBST four times for 5 min each time and finally incubated with substrate. The colored spots were then scanned and quantitated using a densitometer.[14] U266 B1 and RPMI 8226 were cultured at 0.25 × 106/ml with and without 1 mM levamisole for 48 h, and 15 ml of the culture supernatant was collected and lyophilized. The lyophilized samples were reconstituted in 1 ml of PBS and dialyzed. Electrophoresis was carried out on 30 μl of the dialyzed samples. SDS-PAGE was performed according to the method of Laemmli et al. 1970. The gels were silver stained according to the method of Blum et al. 1987.[15] The intracellular IgE content was quantified by western blot assay. Control and levamisole treated cells, 1 × 106, were harvested at 48 h of culture. The cells were centrifuged and washed twice with isotonic saline. The cell lysates were prepared by lysing untreated and levamisole-treated U266 B1 cells. Lysate was separated on a 7.5% SDS-PAGE, and after electrophoresis, proteins on the gel were transferred to nitrocellulose membrane and probed with monoclonal anti-human IgE antibody ALP conjugate. The immunodot blot assay was developed using the substrate BCIP/NBT.


  Results Top


Effect of levamisole on proliferative response and alkaline phosphatase activity of mitogen-stimulated murine splenic lymphocytes

Murine splenic lymphocytes were stimulated with B-cell-specific mitogen LPS and the proliferative response was measured by 3H-thymidine incorporation into DNA at 72 h. Levamisole was added at two concentrations, 0.75 and 1.0 mM. The results are presented in [Figure 1]a. The proliferative response was inhibited by levamisole in a dose-dependent manner (P < 0.05 at 0.75 mM conc). Simultaneously, APase activity of mitogen-stimulated and unstimulated cultures was measured. The results are presented in [Figure 1]b. APase activity was also significantly inhibited by levamisole (P < 0.05).
Figure 1: Effect of levamisole on proliferative response, alkaline phosphatase activity of lipopolysaccharide-stimulated murine splenic B lymphocytes (48-h culture period) (a) proliferative response (b) alkaline phosphatase activity. Values presented are mean ± standard error of the mean of 3 experiments. *Values significantly differ from the respective control, P < 0.05. (1) Control, (2) Lipopolysaccharide 10 μg, (3) Lipopolysaccharide + 500 μM Lev, (4) Lipopolysaccharide + 750 μM Lev, (5) Lipopolysaccharide + 1. 0 mM Lev P < 0.05, 2 versus 4 and 5

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Effect of levamisole on immunoglobulin secretion

LPS-activated B-lymphocytes differentiate into antibody-secreting cells and secrete IgM. The IgM secreted into the cell culture supernatant was estimated using a dot blot assay using a polyclonal anti-IgM antibody (μ-chain specific) conjugated to horseradish peroxidase. The effect of levamisole on IgM secretion by LPS-stimulated murine splenic B-cells was studied and the results are presented in [Figure 2]a. The antibody used for the assay was highly specific as it did not show any cross-reactivity with bovine IgM present in FCS or any other protein. The blot was scanned by densitometry and the results are presented in [Figure 2]b. The amount of IgM secreted by LPS-stimulated cells was significantly inhibited by levamisole (P < 0.05). The proliferative response of myeloma cell lines in the presence of levamisole (1 μM–2.5 mM) was studied using3 H-thymidine incorporation into DNA at 72 h. The results are presented in the [Figure 3]a and b. No inhibition of proliferative response was observed up to a 500 μM concentration of levamisole. However, the proliferation was inhibited in a dose-dependent manner from 0.5 to 2.5 mM levamisole.
Figure 2: (a) Dot blot of IgM secreted by lipopolysaccharide-stimulated murine splenic lymphocytes (b) Densitometric analysis of dot blot. In all cases, 5 μl of samples was used. *P < 0.05, 3 versus 5 and 6. Mean ± standard error of the mean of 3 experiments. (1) Fetal calf serum, (2) Culture supernatant - untreated cells, (3) Lipopolysaccharide-treated cells, (4) Lipopolysaccharide-treated cells +500 μM Lev, (5) Lipopolysaccharide-treated cells +750 μM Lev, (6) Lipopolysaccharide-treated cells +1.0 mM Lev

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Figure 3: Effect of levamisole on proliferative response of myeloma cell lines by 3H-thymidine incorporation into DNA (48.72 h pulse) (a) RPMI 8226 (b) B. U266 B1. Control: No levamisole addition. Each value represents the mean ± standard error of the mean of experiments. *values significantly differ from the respective control, P < 0.05

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Effect of levamisole on alkaline phosphatase activity

Both the myeloma cell lines (RPMI 8226 and U266 B1) constitutively express APase activity. In the presence of levamisole, the APase activity was inhibited in a dose-dependent manner. The results are presented in [Figure 4]a and b. U266 B1 myeloma cells secrete IgE, and RPMI 8226 cells secrete λ light chains constitutively into the culture supernatant. IgE secreted by U266 B1 into the culture supernatant was also estimated by dot blot assay using anti-human IgE antibody, and the results are presented in [Figure 5]a. The secretion of IgE was inhibited by levamisole in a dose-dependent manner as assessed by densitometry [Figure 5]b. However, the western blot analysis showed that much of the Ig remained intracellular when levamisole was added. The cell line RPMI 8226 secretes Ig light chains of λ type, and U266 B1 secretes IgE. Both the cell lines were incubated in a serum-free medium, and the culture supernatants were collected and concentrated. The samples were electrophoresed and the results are presented. The stained bands in lane 1 are due to molecular weight marker; lane 2 shows two bands one at 29 KDa due to light chain of IgE and other at 66 KDa due to heavy chain of IgE from untreated cells. Lane 4 shows a decrease in the concentration of IgE light and heavy chains from levamisole-treated U266B1 cells. Lane 5 shows a single band due to λ light chains secreted by untreated RPMI 8226 cells, and lane 6 indicated that secretion of λ light chains is inhibited in levamisole-treated RPMI 8226 cells.
Figure 4: Effect of levamisole on alkaline phosphatase activity of multiple myeloma cell lines (a) U266 B1 (b) RPMI 8226. Control: No levamisole addition. Each value represents the mean ± standard error of the mean of experiments. *values significantly differ from the respective control, P < 0.05

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Figure 5: (a) Dot blot of immunoglobulin E secreted by U266 B1cells (48 h) (b) Densitometric analysis of dot blot. Mean ± standard error of the mean of 5 experiments *values significantly differ from the control, P < 0.05. (1) Fetal calf serum, 5 μl, (2) Culture supernatant, 5 μl, untreated cells, (3) From cells treated with 0.5 mM Lev, (4) 1.0 mM Lev, (5) 2. 5 mM Lev

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  Discussion Top


Mitogens induce lymphocytes to undergo progressive enlargement, nucleotide synthesis, nuclear protein phosphorylation, histone acetylation, and mitosis. This results in transformation of the normally dormant cells to a rapidly proliferating blast-like state. During the early phase of lymphocyte activation, phosphorylation/dephosphorylation reactions and phosphate transport may play a crucial role in progression of cell through cell cycle. APase distribution is ubiquitous among cell types and tissues. In bone, APase is thought to mediate phosphate assimilation.[16],[17],[18] In intestine, APase is assumed to participate in absorption and transport of lipids and nucleotides.[19] APase may play a role in the renal transport of phosphate.[20] A possible role in phosphate binding has also been proposed.[21] It is observed that APase activity is enhanced when murine splenic lymphocytes are stimulated with LPS, a polyclonal B-cell mitogen. LPS induces the phenotypic maturation of pre-B-cells and immature B lymphocytes to the mature B-cell stage and induces mature B-cells to proliferate and differentiate in to antibody-secreting plasma cells in vitro. Earlier studies in murine system have shown that following B-cell activation and differentiation, the enhancement of APase activity was associated with Ig secretion.[22],[23] Increase in APase activity is an integral feature of B-cell activation and differentiation and perhaps aids in the metabolite transport to fulfill the demands of the growing cell. It could also be involved in the Ig transport in B lymphocytes as it was shown to be complexed and secreted out along with IgM. In view of its pleiotropic role, the enhancement of APase activity appears to be a physiological phenomenon in activated B lymphocytes. However, its function in malignant cell was largely unknown. In this part of study, an attempt has been made to explore the role of APase in B-cell differentiation and possible pathophysiological significance in malignancy.

It was demonstrated that APase activity is constitutively present in myeloma cells and mitogen-stimulated splenic lymphocytes; the proliferative response was associated with concomitant increase in the APase activity. The role of APase in Ig secretion by LPS stimulated lymphocytes and myeloma cells. To begin with, we studied the role of APase using mitogen-stimulated murine lymphocytes, where levamisole inhibited the Ig secretion, indicating that APase is involved in Ig secretion. It is hypothesized that APase may be involved in the constitutive proliferation as well as in Ig secretion of myeloma cells. It was observed that murine splenic lymphocytes showed an increase in proliferative response concomitant with an increase in the APase activity and Ig secretion upon mitogenic stimulation. Levamisole significantly inhibited the APase activity when added to the LPS-stimulated cells at the initiation of the culture. In these cells, the proliferative response and Ig secretion were also inhibited in a dose-dependent manner suggesting that inhibition of APase activity results in inhibition of Ig secretion. Recently, it has been demonstrated that APase activity is expressed in a subset of CD19+ BAP+ normal human B-cells.[17] It has also been shown that upon PWM stimulation of human peripheral blood mononuclear cell, a fraction of proliferating B-cells expressed APase immunologically similar to bone cell APase. The expression of APase correlated with Ig secretion.


  Conclusion Top


In the present work, levamisole added at the initiation of the culture had no effect on the proliferation in normal peripheral blood lymphocytes (PBL) even at 1 mM concentration. This could probably be due to the absence of APase activity in these cells. To investigate the role of APase in proliferation and Ig secretion by myeloma cells, the effect of levamisole was studied. It was found that levamisole inhibited the APase activity, proliferation, and Ig secretion in a dose-dependent manner. These results suggest that APase may have a significant role in B-cell proliferation and Ig secretion. A desirable target in any disease would be a factor uniquely present in and associated with the disease. The presence of APase activity with probable pleiotropic functions in malignant human myeloma cells and its absence in normal B-cells makes APase an attractive target for the modulation of proliferation of myeloma cells. The results of the present studies suggest that APase activity could be used as a target to control the proliferation and differentiation of myeloma cells.

Acknowledgements

Ms B. Nageshwari is supported by a fellowship from CSIR, India. The supply of myeloma cells by NCCS, Pune, is gratefully acknowledged.

Source of funding and scientific ethical approval

CSIR funding and the approval was taken from the University of Hyderabad.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Sun A, Wang YP, Chia JS, Liu BY, Chiang CP. Treatment with levamisole and colchicine can result in a significant reduction of IL-6, IL-8 or TNF-alpha level in patients with mucocutaneous type of Behcet's disease. J Oral Pathol Med 2009;38:401-5.  Back to cited text no. 12
    
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Fararh KM, Farid AS, Mohammed IA, Sultan AA. The hepato-Protective effect of stem cells and levamisole against carbon tetrachloride induced liver fibrosis. BVMJ 2016;31:149-57.  Back to cited text no. 13
    
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Sulimenko T, Dráber P. A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants. J Immunol Methods 2004;289:89-95.  Back to cited text no. 14
    
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Narisawa S, Fröhlander N, Millán JL. Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dyn 1997;208:432-46.  Back to cited text no. 16
    
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Zurutuza L, Muller F, Gibrat JF, Taillandier A, Simon-Bouy B, Serre JL, et al. Correlations of genotype and phenotype in hypophosphatasia. Hum Mol Genet 1999;8:1039-46.  Back to cited text no. 17
    
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Hirano K, Iiizumi Y, Hayashi Y, Tanaka T, Sugiura M, Hayashi K, et al. Role of alkaline phosphatase in phosphate uptake into brush border membrane vesicles from human intestinal mucosa. J Biochem 1985;97:1461.  Back to cited text no. 20
    
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Hossain A, Jung LK. Expression of bone specific alkaline phosphatase on human B cells. Cell Immunol 2008;253:66-70.  Back to cited text no. 23
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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