Influence of Culture Medium on Production of Nitric Oxide and Expression ofInducible Nitric Oxide Synthase by Activated Macrophages In Vitro
Received: 02-May-2016 / Accepted Date: 14-Jun-2016 / Published Date: 19-Jun-2016 DOI: 10.4172/2576-3881.1000108
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
6275Commentary
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 | |||||
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
- Biswas SK, Mantovani A (2014) Macrophages: Biology and role in the pathology of diseases. Springer New York
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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