alexa Influence of Culture Medium on Production of Nitric Oxide and Expression ofInducible Nitric Oxide Synthase by Activated Macrophages In Vitro

ISSN: 2576-3881

Journal of Cytokine Biology

  • Commentary   
  • J Cytokine Biol 2016, Vol 1(2): 108
  • DOI: 10.4172/2576-3881.1000108

Influence of Culture Medium on Production of Nitric Oxide and Expression ofInducible Nitric Oxide Synthase by Activated Macrophages In Vitro

Fumio Amano*
Laboratory of Biodefense & Regulation, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1054, Japan
*Corresponding Author: Fumio Amano, Laboratory of Biodefense & Regulation, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1054, Japan, Tel: 81359271020, Fax: 81359521600, Email: [email protected]

Received Date: May 02, 2016 / Accepted Date: Jun 14, 2016 / Published Date: Jun 19, 2016

Abstract

Activated macrophage phenotypes were influenced by the culture medium; a murine macrophage-like cell line, J774.1/JA-4, expresses different activated-macrophage phenotypes induced by lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ) when the cells are incubated in either Ham’s F-12 medium (F-12) or Dulbecco’s modified Eagle medium (DMEM). Among these phenotypes, NO production and iNOS expression are the most remarkably influenced by the medium; the induction of iNOS mRNA and iNOS protein is higher in DMEM than in F-12, but NO production by activated macrophages is less in DMEM than in F-12. These results suggest that the interpretation of the experimental results requires consideration of the possibility that the differences obtained by different laboratories were caused by the culture medium used.

Keywords: Macrophage activation, Culture medium, Ham’s F-12 medium, Dulbecco’s modified Eagle medium, iNOS, NO production

Commentary

Macrophages play important roles in biology and pathology [1] including those in innate immune responses to pathogens, tumor cells, and apoptotic cells of the host [2-5]. Macrophages also have a unique phenotype, known as “macrophage activation,” which refers to changes their properties in response to pathogen-associated molecular patterns (PAMPs), various cytokines, hormones, and other factors acting as both endogenous and exogenous stimuli, which changes occur through activation processes [1,6,7]. Among activated-macrophage phenotypes, the production of reactive oxygen species (O2- and H2O2), nitric oxide (NO), and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) [8-11] is the major, as well as an important, function of macrophages to exert pivotal roles in the body and to maintain homeostasis [5]. Much research on macrophage has been done by culturing primary macrophages and macrophage-like cell lines in various culture media in vitro . Although there have been some reports describing the effect of different culture media on the cell proliferation and differentiation of macrophages [12-14], no results have been reported precisely concerning the effects of different culture media on macrophage activation.

In a recent report [15], we showed that a murine macrophage-like cell line, J774.1/JA-4, expresses different activated-macrophage phenotypes induced by lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ) when the cells are incubated in either Ham’s F-12 medium (F-12) or Dulbecco’s modified Eagle medium (DMEM). For example, the production of NO, TNF-α, and IL-1β is increased more in DMEM than in F-12 after incubation of the cells continuously for 20 h; whereas the LPS-induced O2- –generating activity is higher in F12 than in DMEM. Besides, after precise study on the mechanisms underlying the induction and expression of inducible NO synthase (iNOS) and its activity, we found that NO production and iNOS expression are the most remarkably influenced by the medium used: the induction of iNOS mRNA and iNOS protein is higher in DMEM than in F-12, but NO production by activated macrophages is less in DMEM than in F-12. iNOS is the key enzyme for the production of NO during inflammation; and it is induced especially by macrophage activation with LPS+IFN-γ, to produce NO from L-arginine (L-Arg), O2, and NADPH as substrates [16]. Concerning Ca2+, iNOS is Ca2+/ calmodulin independent, unlike endothelial NOS (eNOS); but there is a report that elevated intracellular calcium affects NO production by iNOS [17]. Comparing the chemical compositions of these media (Table 1), F-12 contains a 2.5 times higher amount of L-Arg than DMEM, whereas the latter contains a 6.0 times higher amount of Ca2+ than the former. Although it seems feasible that the difference in NO production might have been caused by the differences in L-Arg and Ca2+ levels in these media, our preliminary results showed that the addition of L-Arg to DMEM or that of Ca2+ to F-12 to adjust the concentration of each to be equal to that in the other medium failed to influence NO production from activated macrophages (data not shown). Aside from the differences in L-Arg and Ca2+, DMEM contains higher amounts of glucose and phenol red; whereas F-12 contains higher ones of pyruvate and vitamins (Table 1), both of which have been reported to have some influence on iNOS activity [18,19]. However, none of them have been shown to have a noticeable effect on the expression of iNOS or production of NO (data not shown). It should be also noted that macrophages show a significantly reduced level of NADPH, a substrate of iNOS as well as NADPH oxidase, during incubation in DMEM [15], which reduction might lower iNOS activity.

    F-12 11765 DMEM 11965 #1 Ratio #1 Ratio
Components M.W mg/L mM mg/L mM F-12/DMEM(mM) $ Ratio DMEM/F-12(mM) $Ratio
Amino Acids                  
Glycine 75 7.5 0.1 30 0.4 0.25   4 *
L-Alanine 89 8.9 0.1         0  
L-Arginine hydrochloride 211 211 1 84 0.398 2.51 * 0.4  
L-Asparagine-H2O 150 15.01 0.1         0  
L-Aspartic acid 133 13.3 0.1         0  
L-Cysteine hydrochloride-H2O 176 35.12 0.2         0  
L-Cysteine 2HCl 313     63 0.201 0      
L-Glutamic Acid 147 14.7 0.1         0  
L-Glutamine 146 146 1 584 4 0.25   4 *
L-Histidine hydrochloride-H2O 210 21 0.1 42 0.2 0.5   2 *
L-Isoleucine 131 4 0.031 105 0.802 0.04   26.25 **
L-Leucine 131 13.1 0.1 105 0.802 0.12   8.02 *
L-Lysine hydrochloride 183 36.5 0.199 146 0.798 0.25   4 *
L-Methionine 149 4.5 0.03 30 0.201 0.15   6.67 *
L-Phenylalanine 165 5 0.03 66 0.4 0.08   13.2 **
L-Proline 115 34.5 0.3         0  
L-Serine 105 10.5 0.1 42 0.4 0.25   4 *
L-Threonine 119 11.9 0.1 95 0.798 0.13   7.98 *
L-Tryptophan 204 2.04 0.01 16 0.078 0.13   7.84 *
L-Tryosine disodium salt dihydrate 262 7.81 0.03 104 0.398 0.07   13.37 **
L-Valine 117 11.7 0.1 94 0.803 0.12   8.03 *
Vitamins                  
Biotin 244 0.007 0.00003         0  
Choline chloride 140 14 0.1 4 0.029 3.5 * 0.29  
D-calcium pantothenate 477 0.5 0.001 4 0 0.13   8 *
Folic Acid 441 1.3 0.003 4 0.009 0.32   3.08 *
Niacinamide 122 0.036 0.0003 4 0.033 0.01   111.11 ***
Pyridoxine hydrochloride 206 0.06 0.0003 4 0.019 0.02   66.67 **
Riboflavin 376 0.037 0.0001 0.4 0.001 0.09   10.81 **
Thiamine hydrochloride 337 0.3 0.001 4 0.012 0.08   13.33 **
Vitamin B12 1355 1.4 0.001         0  
i-Inositol 180 1.8 0.1 7.2 0.04 2.5 * 0.4  
Inorganic Salts                  
Calcium Chloride (CaCl2) (anhyd.) 111 33.22 0.299 200 1.802 0.17   6.02 *
Cupric sulfate (CuSO4-5H2O) 250 0.003 0.00001         0  
Ferric sulfate (FeSO4-7H2O) 278 0.834 0.003         0  
Ferric Nitrate (Fe(NO3)3"9H2O) 404     0.1 0.0002 0      
Magnesium Chloride (MgCl2) (anhydrous) 95 57.22 0.602         0  
Magnesium Sulfate (MgSO4) (anhyd.) 120     97.67 0.814 0      
Potassium Chloride (KCl) 75 223.6 2.981 400 5.333 0.56   1.79  
Sodium Bicarbonate (NaHCO3) 84 1176 14 3700 44.048 0.32   3.15 *
Sodium Chloride (NaCl) 58 7599 131.017 6400 110.345 1.19   0.84  
Sodium Phosphate monobasic (NaH2PO4-H2O) 138     125 0.906 0      
Sodium Phosphate dibasic (Na2HPO4) anhydrous 142 142 1         0  
Zinc sulfate (ZnSO4-7H2O) 288 0.863 0.003         0  
Other Components                  
D-Glucose (Dextrose) 180 1802 10.011 4500 25 0.4   2.5 *
Hypoxanthine Na 159 4.77 0.03         0  
Linoleic Acid 280 0.804 0.0003         0  
Lipoic Acid 206 0.21 0.001         0  
Phenol Red 376.4 1.2 0.003 15 0.04 0.08   12.5 **
Putrescine 2HCl 161 0.161 0.001         0  
Sodium Pyruvate 110 110 1         0  
Thymidine 242 0.7 0.003         0  
(Data modified from Gibco, Thero Fisher Scientific catlog) ($ Remarkable Ratios are shown as * >2.0, ***>10.0, and ***>100.0.)

Table 1: Chemical compositions of F12 and DMED medium, and ratios of them.

Further study is necessary to determine which component of the chemical composition of these media is responsible for the altered macrophage phenotypes. We are now testing the effects of each component in these media on mouse peritoneal macrophages and RAW264.7 macrophage-like cells, especially with special interest regarding those which are at more than 2-, 10- or even 100-fold higher concentrations in one medium versus the other, as shown in Table 1.

Other things deserving comment are the following two: One is the importance of our findings [15] regarding performing experiments with different media. The cumulative literature concerning macrophage activation is based mainly on individual studies of researchers who have used different culture medium such as F-12, DMEM, or RPMI1640 in their macrophage-activation experiments. Therefore, the interpretation of their results seems to require consideration of the possibility that the differences obtained by different laboratories were caused by the culture medium used.

The other is the importance of our findings [15] with respect to future studies. Macrophages are located in a variety of tissues and organs as monocytes (progenitors of macrophages), tissue-specific macrophage-like Kupffer cells (liver), microglia cells (brain), alveolar macrophage (lung) [1], and also in tumors or inflammatory lesions in response to pathological stimulations. These varied distributions of macrophages seem to suggest the possibility of their exposure to different nutritional components in their micro-environments, leading to altered activation phenotypes of the macrophages. Through our study, we hope to obtain further new findings concerning the chemical components having remarkable influence on macrophage activation.

References

Citation: Amano F (2016) Influence of Culture Medium on Production of Nitric Oxide and Expression of Inducible Nitric Oxide Synthase by Activated Macrophages In Vitro. J Cytokine Biol 1:108. Doi: 10.4172/2576-3881.1000108

Copyright: © 2016 Amano F. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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