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Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 69 

Original Article 

Adenosine receptor agonists suppress macrophage inflammatory protein -1α 

production and collagen induced arthritis  

Hari Sonwani, Virendra Kumar Sharma, Steffi Thomas, Akhlesh Kumar 

From, Department of Pharmacology, School of Pharmacy, LNCT University, Kolar Road ,Bhopal 462042 [M.P] India. 

Correspondence to: Hari Sonwani, Department of Pharmacology, School of Pharmacy, LNCT University, Kolar Road, Bhopal-

462042 Madhya Pradesh, India. Email: harisonwani10@gmail.com  

ABSTRACT 

The production of pro- and anti-inflammatory cytokines is modulated by ligands of various adenosine receptor subtypes. In this study, 

we looked at how adenosine and different ligands for the adenosine receptor subtypes (A1, A2, A3) affected the production of the 

chemokine macrophage inflammatory protein (MIP) 1a in immune-stimulated RAW macrophages in vitro. In addition, we 

investigated whether an A3 adenosine receptor agonist reduces MIP-1α production and influences the course of inflammation in 

collagen-induced arthritis. The A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and, less 

potently, the A2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylamino-5'-N-ethylamino-5'-N- ethylamino-5'-N 

Adenosine was a mild inhibitor, and the selective A1 receptor agonist 2-chloro- N-cyclopentyladenosine (CCPA, 1 200 M) proved 

ineffective. MIP-1α production is inhibited by suppression of its steady-state mRNA levels and was linked to the A3 and A2 agonists. 

We concluded that stimulation of A3 and, to a lesser extent, A2 adenosine receptors decreases MIP-1α expression based on our in 

vitro findings. We studied whether IB-MECA, as the most powerful inhibitor of MIP-1α expression, also influences the production of 

other pro-inflammatory mediators. IB- MECA (1 300 M) reduced the production of IL-12, IL-, and, to a lesser extent, nitric oxide in 

immune-stimulated cultured macrophages in a dose-dependent manner. We explored whether the A3 agonist IB-MECA alters the 

course of inflammation, MIP-a production, and the degree of neutrophil recruitment in arthritis because MIP-α is a chemokine that 

increases neutrophil recruitment into inflammatory areas. In the case of IB- MECA (0.5 mg/kg/day) decreased the degree of joint 

inflammation in a mouse model of collagen-induced arthritis. IB-MECA decreased neutrophil infiltration and inhibited the production 

of MIP-1α, IL-12, and nitrotyrosine (a marker of reactive nitrogen species) in the paws. We conclude that adenosine receptor agonists, 

particularly the A3 agonist IB-MECA, inhibit MIP-α synthesis and have anti-inflammatory properties. As a result, stimulating 

adenosine receptor subtypes A3 and A2 may be a promising technique for the treatment of acute and chronic inflammatory diseases. 

Key words: Inflammation, Cytokines, Adenosine Receptor, Arthritis 

denosine is a purine nucleoside generated by a 

number of cells in response to metabolic stress 

Adenosine receptor ligands A1, A2a, A2b, and A3 

have been found to inhibit the synthesis of pro-inflammatory 

mediators, diminish the recruitment of polymorphonuclear 

leukocytes (PMNs), and exhibit anti-inflammatory properties 

[1]. Furthermore, the ability to release adenosine is related to 

the anti-inflammatory activitie of medicines such methotrexate, 

sulphasalazine, and adenosine kinase inhibitors Both A1 and 

A2 receptor agonists were reported to decrease TNF- α 

production in RAW 24.7 cells in recent research macrophages 

or human monocytes, and the rank order of agonist potency 

was not typical of either A1 or A2 receptors implying that the 

A3 receptor may be involved. Furthermore, adenosine, but not 

adenosine, in human monocytes, selective A1 and A2 receptor 

agonists increased IL-10 production It was further proven that 

suppression of TNF-α generation by LPS-stimulated U937 

(human monocyte) cells was primarily an A3 receptor- 

mediated mechanism using particular A1, A2 and A3 

receptor-mediated mechanism using particular A1, 

A2, and A3 receptor agonists and antagonists in the murine 

J774.1 macrophage cell line, ADO receptor agonists 

prevented endotoxin activation of the TNF- α gene and protein 

expression in a dose-dependent manner similar to the A3 

receptor [2].  

 Although the above research showed that several 

adenosine receptor agonists can affect the production of pro- 

and anti-inflammatory cytokines no information on the 

modification of the production of chemokines by adenosine 

receptor ligands was previously published. MIP-1 α 

(macrophage inflammatory protein) is a low-molecular-weight 

CC chemokine that has significant inflammatory effects [3]. 

The enhancement of neutrophil recruitment is the key 

mechanism for inflammatory sites. We investigated whether 

adenosine receptor ligands affect the production of MIP-1 α 

A 

mailto:harisonwani10@gmail.com


Sonwani et al.                                                                Role of adenosine receptor agonists in inflammatory conditions 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 70 

protein and mRNA in macrophages because MIP-1 α is an 

important mediator of acute inflammation anadenosine 

receptor ligands can exert potent anti-inflammatory effects. 

We investigated whether the A3 agonist IB-MECA impacts 

MIP-1α production, the course of inflammation, and neutrophil 

recruitment in a model of collagen-induced arthritis after 

finding that adenosine receptor agonists, most notably the A3 

agonist, suppress MIP-1α expression. 

METHODS 

Adenosine agonists have an effect on MIP-la production in 

RAN 264.3 cells: The mouse macrophage cell line RAW 24.7 

was grown. 1 mL of 1300 mM adenosine, the A1 receptor 

agonist 2-chloro-N-cyclopentyladenosine (CCPA), the A2 

receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-

ethyl-carboxamidoadenosine (CGS- 2180), and the A3 receptor 

agonist N-(3-iodobenzyl)-adeno. Research Biochemicals Inc. 

provided adenosine agonists. After 30 minutes, cells were 

stimulated with LPS, and supernatants or cells were collected 

1–3 hours later for MIP-1α protein or mRNA assays. The 

effect of a 1-h pretreatment with the adenosine deaminase 

inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, 50 M) 

on the inhibitory effect of adenosine on MIP-1α production 

was investigated in a second set of tests. MIP-1α is a protein 

that was discovered in the human body as previously 

described, using murine ELISA kits bought from Genzyme ) 

(detection limit: 1.5 pg/ml) The experiment was not hampered 

by any test chemicals. 

Adenosine agonists' effects on MIP-la steady-state mRNA 

levels: A guanidinium isothiocyanate/chloroform-based 

approach (TRIZOL) was used to extract total RNA from each 

well, followed by isopropanol precipitation. On a 1% 

formaldehyde gel, cytoplas mic RNA (15 g) was separated and 

transferred to a nylon membrane. The rat MIP-1α cDNA 

sequence is 92 percent identical to the mouse sequence, 

indicating cross-hybridization between the two species. By 

random priming, this cDNA was radiolabeled with 32P-dCTP 

(specific activity, 3,000. The radioactivity of probes was 

measured. The Northern blot was hybridized at 42°C for 1 

hour after scintillation counting and 1.5107 c.p.m. were 

applied. The hybridized filters were washed serially at 53°C 

with a solution of 2 sodium citrate, sodium chloride, and 0.1 

percent SDS (2SSC). Autoradiography was performed using 

Hodak -OMAT-AR film at —70°C. Membranes were stripped 

with boiling 5 mM EDTA and rehybridized with a 32P-

radiolabeled oligonucleotide probe for 18S ribosomal RNA 

after probing for MIP-1α. In RAN 264.3 cells, the effect of IB- 

MECA on cytokine and NO production. 

 In the instance of the A3 agonist IB-MECA, we wanted 

to see if it affected the production of IL- 12, IL-, and NO in 

RAW 24.7 macrophages as well. The cells were pretreated 

with 1 300 MIB-MECA in these tests. After 30 minutes, cells 

were stimulated with either LPS (10 g ml—1) or murine 

IFN- (200 u ml—1; Genzyme, Boston, MA, USA). This LPS 

concentration is higher than that employed in investigations 

on the effects of adenosine receptor agonists on MIP-1α 

generation. Our early research has shown that greater LPS 

concentrations are required to reach optimal NO generation 

levels. Only the combination of a greater dose of LPS and 

IFN- resulted in detectable IL-12 production in this cell type. 

After a 24-hour incubation period at 37°C, the culture 

supernatant fluids were collected and kept at —70°C. ELISA 

kits specifically for murine cytokines was used to determine 

cytokines. 

 ELISA kits from Genzyme were used to quantify IL-12 

(p40 and p70) and IL- A Spectramax 250 microplate reader 

was used to read the plates at 450 nm. IL- 12 (p40) had 

detection limits of 10 pg ml—1, IL-12 (p70) had detection 

limits of 5 pg ml—1, and IL- had detection limits of 5 pg 

ml—1. Assays were carried out as previously described. And 

mg/ml by stirring at 4°C overnight. CII was dissolved and 

stored at —70°C until needed. The addition of 

Mycobacteuium tuberculosis H37Ra at a dose of 2 mg ml—1 

yielded Complete Freund's adjuvant (CFA). CII was 

emulsified with an equal volume of CFA before injection. 

As previously described (Szabo' et al., 1998), collagen-

induced arthritis was developed. On day 1, mice were given 

100 l of the emulsion (containing 100 g CII) intradermally at 

the base of the tail. A second injection of CII in CFA was 

given on day 21. Animals were given IB-MECA at a dose of 

0.5 mg kg—1 to stimulate A3 adenosine receptors. This dose 

was chosen based on prior in vivo investigations Auchampach 

et al., 1997; Tracey et al., 1994; Tracey et al., 1997). Starting 

on day 18, animals were given either vehicle (n=18) or IB- 

MECA (n=18; 0.5 mg kg—1, i.p.) every 24 hours. The mice 

were checked for arthritis on a daily basis. Employing a 

macroscopic scoring system that ranges from 0 to 4 (0=no 

indications of arthritis, 1=swelling and/or redness of the paw 

or one digit, 2=two joints involved, 3=more than two joints 

involved, and 4=severe arthritis of the entire paw and digits). 

Each mouse's arthritic index was computed by adding the 

four unique paw scores. No animals were removed from the 

calculations, and severity indices were determined for entire 

groups of mice (vehicle-treated or IB- MECA-treated). 

Nitrotyrosine immunohistochemistry and histology: 

Animals were sacrificed under anesthesia at the end of the 

experiments (Day 35), and their paws and knees were taken 

and preserved for histological analysis by an investigator who 

was blinded to the treatment regimen. Joints were embedded 

for nitrotyrosine immunohistochemistry. Snap frozen in liquid 

nitrogen in M1 medium. A microtome equipped with a carbide 

steel knife was used to cut cryostat pieces (m). By 

immunohistochemistry, joint slices were examined for the 

presence of nitrotyrosine, a peroxynitrite indicator For 15 

minutes, endogenous peroxidase was quenched with 0.3 



Sonwani et al.                                                                Role of adenosine receptor agonists in inflammatory conditions 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 71 

percent H2O2 in PBS.  By incubating the slice in 2 percent 

normal goat serum in phosphate-buffered saline for 0 minutes, 

non-specific adsorption was reduced. After that, the sections 

were treated with a 1: 500 dilution of primary anti-

nitrotyrosine antibody overnight biotin-conjugated goat anti-

rabbit IgG, and an avidin-biotin-peroxidase complex was used 

to identify specific labeling. Anti-nitrotyrosine antibodies 

were used to incubate sections in control studies. In the 

presence of 10 mM nitrotyrosine, an antibody was produced. 

The nitrotyrosine staining in the figures was removed as a 

result of this intervention. 

In paw extracts, cytokines, chemokines, and nitrotyrosine 

were detected: Aqueous joint extracts were prepared from 

control animals and animals with 35 days of arthritis as 

described by homogenization in a lysis buffer in the presence 

of a mixture of protease inhibitors (10 g ml—1 aprotinin, 20 g 

ml—1 leupeptin, 10 g ml—1 pepstatin A, and 1 mM PMSF, 

pH 7.25) ELISA ) was used to determine the presence of IL-

12, TNF-α, and MIP-1α in the extracts, as well as the presence 

of nitrated proteins using Western blotting Each sample 

weighed thirty gram heated to 95°C for 3 minutes after being 

diluted in an equal volume of treatment buffer. After that, the 

samples were put into a 1% Tris-Glycine solution. Gels were 

operated for 2 hours at 120 volts, then transferred to 0.45 m 

nitrocellulose at 30 volts for 0 minutes using the 1/2Towbin 

buffer system (1.45 g Tris, 7.2 g glycine, 800 ml di H2O and 

200 ml MeOH). The membrane was blocked in 1 percent BSA: 

1 percent nonfat milk in PBS-Tween (phosphate-buffered 

saline with 0.05 percent Tween 20) for 1 hour before being 

probed with rabbit anti-nitrotyrosine (1 g/ ml in PBS- Tween) 

overnight at 4°C. The blot was washed three times with PBS-T 

and once with H2O before being incubated with the secondary 

antibody, goat anti-rabbit-HRP, for 1.5 hours (1 : 3000). After 

washing the blot three times in PBS-T and once in di H2O, 1.5 

ml mixed ECL chemiluminescence reagent was applied for 1 

minute. After that, the blot was exposed to X-ray film for 0 

seconds. 

Measurements of myeloperoxidase in paw homogenates: In 

addition, the activity of myeloperoxidase, a marker of 

neutrophil infiltration, was evaluated in the paws as described 

Paws were homogenized in a solution of 0.5 percent hexa-

decyl-trimethyl- ammonium bromide mixed in 10 mM 

potassium phosphate buffer (pH 7) and centrifuged at 20,000g 

for 30 minutes at 4°C. An aliquot of the supernatant was 

allowed to react with tetra- methyl-benzidine (1. mM) and 0.1 

mM H2O2 in a solution. At a wavelength of 50 nm, 

spectrophotometry was used to determine the rate of change in 

absorbance. Myeloperoxidase. The amount of enzyme that 

degraded 1 mol of hydrogen peroxide per minute at 37°C was 

measured in milliunits per mg protein. 

Analyzing and presenting data: All figures and text data are 

expressed as the mean of n observations, where n is the 

number of wells (9 wells from two to three independent 

experiments) or the number of animals analyzed. One- and 

two-way analysis of variance were used to investigate the data 

sets, and individual group averages were compared using 

Dunnett's test. The statistical differences in the arthritic 

indices were tested using the Mann-Whitney U-test (2-tailed, 

independent) in the arthritis studies. Statistical significance 

was defined as a P-value of less than 0.05. 

RESULTS 

MIP-l α is suppressed by adenosine agonists in immune-

stimulated macrophages.MIP-1α was produced in 

considerable amounts in response to LPS (10 ng/ml). The A3 

agonist IB-MECA and, to a lesser extent, the A2 agonist 

CGS-2180 inhibited MIP-1α production, whereas the A1 

receptor agonist CCPA had no impact (Figure 1). At 200 M, 

adenosine had a minor inhibitory effect, resulting in a 213 

percent inhibition (P<0.01). The MTT experiment revealed 

that the adenosine agonists tested had no effect on 

mitochondrial respiration. Because adenosine deaminase 

degrades adenosine quickly, we wanted to see if inhibiting 

adenosine deaminase changed the degree of inhibition. There 

was no increase in the inhibitory action of adenosine in the 

presence of the adenosine deaminase inhibitor EHNA (50 M) 

(245% inhibition at 200 MMIP-1α steady-state mRNA levels 

rise in a time-dependent manner IB-MECA caused a 

significant reduction in MIP-1α production, which was 

accompanied by a significant, dose-dependent reduction in 

MIP-1α mRNA (Figure 2). MIP-1α mRNA expression was 

unaffected by adenosine or the A1 agonist, however, steady-

state mRNA levels were decreased by the A2 agonist CGS-

2180 (Figure 2).  

Overall, the degree of inhibition of MIP-1α protein and 

MIP-1α mRNA expression by adenosine agonists had a 

fairly high association. The degree of suppression of MIP-1α 

mRNA levels and protein levels, on the other hand, did not 

always match. Using the ELISA approach, we discovered 

that CGS (at 200 M) suppressed MIP-1α protein synthesis by 

roughly 50% whereas the inhibition of MIP-1α protein 

production by nearly 50% (Figure 1) LPS elicited the 

synthesis of mRNA was virtually completely eliminated 

Differences in measurement duration (protein was tested 3 

hours after LPS, mRNA 2 hours after LPS) and/or sensitivity 

of the two assays. These disparities could be due to the 

methods utilized (ELISA versus Northern blotting).  

IB-MECA inhibited MIP-1α production the most strongly 

of all the adenosine agonists tested. As a result, we examined 

the effect of this agonist on the generation of other 

inflammatory mediators (IL-12, IL-, NO) by activated 

macrophages and in an in vivo model of inflammation in the 

following experiments. LPS (10 g ml—1) or IFN- (200 u 

ml—1) did not elicit measurable levels of IL-12 (p40 or p70) 



Sonwani et al.                                                                Role of adenosine receptor agonists in inflammatory conditions 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 72 

in RAW 24.7 macrophages after a 24-hour treatment Even 

though the combination of LPS (10 g ml—1) and IFN- (200 

u ml—1) promoted the production of IL-12 p40 (Figure 4a), 

IL-12 p70 was not generated in measurable amounts. 

 Pretreatment of cells with IB-MECA 30 minutes before 

LPS + IFN- resulted in a concentration- dependent reduction 

of IL-12 p40 production, as measured at 24 hours (Figure 4a). 

As determined 24 hours after stimulation, LPS (10 g ml—1) 

stimulated the release of IL- and nitrite (the breakdown 

product of NO) in the culture supernatants of RAW 24.7 cells. 

IB-MECA, administered 30 minutes before LPS, reduced the 

generation of IL- and nitrite (Figure 4b and c). In all of these 

trials, IB-MECA had no effect on cell viability as assessed 

by the MTT assay (not shown). IB- stronger MECA's 

inhibition of IL-12 and MIP-1α and less reduction of NO and 

IL- production could be due to two distinct A3 receptor 

subtypes. The recent research, on the other hand. 

IB-MECA therapy protects mice from collagen-induced 

arthritis: The majority of the vehicle-treated mice 

developed arthritis between days 2 and 35 after the first 

collagen immunization Treatment with IB-MECA (0.5 

mg/kg once day i.p.) reduced the occurrence of arthritis and 

lowered the disease's severity. The paws of the vehicle-

treated arthritic animals showed evidence of severe 

suppurative arthritis at day 35, with extensive neutrophil, 

macrophage, and lymphocyte infiltration. In addition, there 

was severe or moderate necrosis, hyperplasia, and sloughing 

of the synovium, as well as inflammation spreading into the 

neighboring musculature The degree of arthritis was 

dramatically reduced in the IB-MECA treated mice, with 

moderate cell infiltration, mild to moderate necrosis, and 

synovial hyperplasia The reduction in neutrophil infiltration 

was measured by paw myeloperoxidase.  

Arthritis induced a 991 mU/mg protein increase in 

myeloperoxidase levels (n=12). There was a significant 

increase in the number of IB-MECA-treated animals. At the 

end of arthritis, there was a substantially decreased degree of 

rise in the paw myeloperoxidase concentration (3 mU/mg 

protein; n=12, P0.01). At 35 days, there was a considerable 

increase in MIP-1α and IL-12 p40 levels in the aqueous 

extracts of the arthritic paws, but no detectable TNF-α or IL-

12 p70 levels in the extracts IB- MECA therapy reduced the 

formation of LPS-stimulated cells or LPS-challenged 

animals, according to the results in LPS-stimulated cells or 

LPS-challenged animals. Increased levels of IL- 12 p40 and 

MIP-1α in the joints (Figure 7). We detected the emergence 

of nitrotyrosine-positive staining in the inflamed joints using 

immunohistochemistry and Western blotting of proteins in 

aqueous joint extracts, but not in healthy control animals. 

The degree of nitrotyrosine staining was lowered after IB-

MECA therapy (Figure 3, 4 & 5). 

 
Figure 1 (a): Effect of adenosine [ADO], the A1 agonist 

CCPA, the A2 agonist CGS, and the A3 agonist IB-

MECA on the production of MIP-1α mRNA in response 

to stimulation with bacterial lipopoly-saccharide LPS (10 

ng ml—1) in RAW macrophages. (a) Dose-response 

showing the effect of 0.5, 5, 50, and 100 μM IB-MECA on 

the production of MIP-1α mRNA at 3 h after stimulation. 

(b) Effect of adenosine, CCPA, CGS, and IB-MECA (200 

μM each) on the production of MIP-1α mRNA at 3 h 

after stimulation. Representative blots of n=3 − 4 blots 

are shown; 18S mRNA was unaffected by any of the 

treatments were. This possibility was not intended to be 

explored. 

 

Figure 2: Effect of adenosine [ADO], the A1 agonist 

CCPA, the A2 agonist CGS, and the A3 agonist IB-

MECA on the production of MIP-1α at 3 h after 

stimulation with bacterial lipopolysaccharide LPS (10 ng 

ml—1) in RAW macrophages. MIP-1α production in the 

absence of the inhibitors amounted to б.б±1.3 ng ml—1, 

and was considered 100%. n=б − 9 wells from two to 

three independent experiments. *P<0.05 and **P<0.01 

indicate significant inhibition of the production of MIP-

1α. 



Sonwani et al.                                                                Role of adenosine receptor agonists in inflammatory conditions 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 73 

 
Figure 3 – (a) IB-MECA suppresses collagen-induced 

arthritis in mice. The percentage of arthritic mice (mice 

showing clinical scores of arthritis >1) is represented. (b) 

Effect of IB-MECA on the severity of collagen-induced 

arthritis. The median arthritic score during collagen-

induced arthritis. n=10 − 12. There was a significant 

increase in the arthritic score from day 28 (*P<0.01), and 

there was significant suppression of the arthritic score by 

IB-MECA from day 30 (¢P<0.05). 

 
Figure 4: IB-MECA suppresses the production of 

inflammatory mediators in immune-stimulated cultured 

RAW macrophages. Cells were stimulated with LPS (10 

μg ml—1) and IFN-ц (200 mU/ml) for 24 h for the 

measurement of IL-12 (p40), with LPS (10 μg ml—1) for 

24 h for IL-б and nitrite measurements, or with LPS (10 

ng/ml) and IFN-ц(200 mU ml—1) for 3 h for the 

measurement of MIP-1α Absolute values of IL-12, IL-б, 

and nitrite in the absence of IB- MECA treatment 

amounted to 378±23 pg ml—1, 7б±4 ng ml—1 and 49±5 

μM respectively, and were considered 100%. n=б − 9 

wells from two to three independent experiments. 

*P<0.05 and **P<0.01 indicate significant inhibition by 

IB-MECA treatment.  

 

Figure 5 - IB-MECA suppresses the production of 

inflammatory mediators in aqueous paw extracts of mice 

subjected to collagen-induced arthritis. Levels of MIP-1α 

and IL-12 are shown in samples from control animals 

(C), from collagen-induced arthritis at 35 days (A), and 

IB-MECA-treated mice subjected to collagen- induced 

arthritis (M) at 35 days. n=10 − 12. *P<0.05 indicates 

suppression of IL-12 and MIP-1α production in arthritis 

by IB- MECA.  

DISCUSSION 

Different adenosine receptor subtypes activate different 

signal transduction processes in different cells. Martin and 

Dorf (1991) In macrophages, however, A3 receptor 

stimulation does not require cyclic AMP, protein kinase A, 

or the transcription factor NF-kappa B, despite the fact that 

stimulation of this receptor subtype might change the 

makeup of the AP-1 transcription complex Further research 

is needed to determine the cellular signaling pathway by 

which A3 activation reduces cytokine or MIP-1α production 

[3-5]. In our settings, the A1 agonist had no substantial 

inhibitory effect on MIP-1α generation, but adenosine was a 

weak inhibitor. Adenosine is primarily an A1 receptor 

agonist, with minor A2 and A3 agonist properties [6]. Our 

research with the adenosine deaminase inhibitor showed that 

adenosine deaminase, which is present in the fetal calf, 

degrades adenosine quickly. The low amount of inhibition is 

not attributable to serum. The lower eAcacy of adenosine in 

MIP-1α production may be due to its reduced potency on A2 

and A3 receptors, according to our findings [7]. 

 In addition to inhibiting MIP-1α production, IB-MECA 

decreased IL-12, IL-, and NO production in immune 

stimulated cultured macrophages, according to the current 

study. Adenosine receptor agonists have recently been 

shown to prevent endotoxin-mediated stimulation of the 

TNF-α gene and protein expression in the murine J774.1 

macrophage cell line in a dose-dependent manner 

characteristic of the A3 receptor [9, 10].  We recently 

demonstrated that stimulating A3 receptors with the selective 

A3 receptor agonist IB-MECA reduced plasma TNF-α while 



Sonwani et al.                                                                Role of adenosine receptor agonists in inflammatory conditions 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 74 

increasing IL-10 in LPS-treated mice. The current study's 

findings add to these earlier findings. The RAW 24.7 cell 

studies are merely a rough approximation of the properties of 

primary or resident macrophages, or the in vivo scenario in 

arthritis, where resident macrophages play a role in the 

production of chemokines, cytokines, and NO [11].  

 Despite this, The control of chemokine, cytokine, and 

NO production by adrenergic, purinergic receptors in 

monocytic/macrophage cells and primary monocytes/ 

macrophages displays striking parallels The in vivo 

assessments of NO, IL-12, and MIP-1α production in joint 

extracts in the current investigation were similarly in good 

agreement with our in vitro findings in RAW cells. In the 

arthritis model, it's possible that IB- MECA inhibits the 

synthesis of IL-12, MIP-1α, and NO, which are all significant 

anti-inflammatory mechanisms [12-14]. It is widely known 

that IL-12 plays a key role in the early events of the arthritis 

induction phase. The research best confirmed this, showing 

that IL-12 may substitute mycobacteria and cause 

autoimmune arthritis in DBA/1 mice inoculated with chicken 

CII in incomplete Freund's adjuvant [15]. Furthermore, these 

researchers discovered that IL-12 could aggravate arthritis 

caused by chicken CII in CFA mice. The course of the 

disease was suppressed in IL-12 deficient animals or mice 

treated with anti-mIL-12 antibodies [13].  

 After IL-12 initiates the inflammatory process, 

macrophages and other cell types become activated and 

release a range of mediators such as TNF- a, IL-, IL-1, MIP-

1α, and NO, which keep the inflammatory state alive. The 

chemokine MIP-1α, which is generated by fibroblasts or 

macrophages, appears to be a key pathogenic element in the 

development of arthritis, owing to its chemotactic action on 

inflammatory cells. The decrease of paw MIP-1α levels may 

contribute to the reduction of neutrophil recruitment (as 

evidenced by the suppression of paw myeloperoxidase levels 

in the current investigation) [14-16]. The A3 has an inhibiting 

action. Over production of this free radical, as well as its 

reactive reaction product, peroxynitrite, has been 

demonstrated to contribute to the pathogenesis of the 

inflammatory joint disease, therefore a receptor agonist on 

NO release could be advantageous Although recent research 

postulated alternate mechanisms of tyrosine nitration, 

connected to myeloperoxidase-dependent conversion of 

nitrite to NO2Cl and NO2, nitrotyrosine production is largely 

acknowledged as a specific 'footprint' of peroxynitrite In the 

joints, nitrotyrosine could operate as a collective indication 

for the formation of reactive nitrogen species [17-20]. 

 The current work shows that stimulating the A3 and A2 

receptors reduces MIP-1α production.by inhibiting the 

expression of its mRNA Furthermore, the current study 

shows that an A3 receptor agonist reduces inflammation in 

collagen-induced arthritis. Downregulation of the pro-

inflammatory mediators MIP-1α, IL-12, and NO is one of the 

mechanisms underlying this anti-inflammatory action. The 

ability of drugs like methotrexate sulphasalazine and 

adenosine kinase inhibitors to release adenosine at the sites 

of inflammation is linked to their anti-inflammatory 

properties. These anti-inflammatory actions may be related to 

A3 and A2, at least in part, according to our hypothesis of 

activation of receptors. We believe that stimulating the 

adenosine receptor subtypes A3 and A2 could be a promising 

technique for the treatment of acute and chronic 

inflammatory diseases. 

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How to cite this article: Hari Sonwani, Virendra Kumar 

Sharma, Steffi Thomas, Akhlesh Kumar. Adenosine 

receptor agonists suppress macrophage inflammatory 

protein -1α production and collagen induced arthritis. 

Indian J Pharm Drug Studies. 2023: 2(2) 69-75. 

Funding: None               Conflict of Interest: None Stated 

 


