**6. Effect of μOR activation with a selective ΚOR agonist and μOR antagonist**

The effect of coactivation of two different opioid receptors, U50488, a selective KOR agonist was used for lymphocyte activation, and effect of costimulation of μOR was achieved by using DAMGO, an opioid receptor agonist. Cells were treated with μOR agonists DAMGO for varying time intervals, and effect on cell proliferation and NF-κΒ phosphorylation was studied. Results using HepG2 revealed that U50488 (an agonist for KOR) was able to mitigate the effect of DAMGO, an agonist for μOR, and a significant reduction in the percentage of cells in both M-phase and S-phase of the cell cycle was observed at 2 and 24 h of treatment. U50488 alone also showed a larger percentage of cells in G0 phase both at 2 and 24 h in comparison with DAMGO, and the mitigation of U50488 induced effects indicating cross talk of receptors (**Figure 3**).

**91**

treatment (**Table 4**).

**Figure 3.**

*phosphorylation.*

U50488+ DAMGO

*DAMGO vs U50488.*

*DAMGO vs U50488 + DAMGO.*

*U50488 vs U50488+ DAMGO.*

*1.4*

*2.5*

*3.6*

**Table 4.**

dissociation [90].

*Immune Cell Activation: Stimulation, Costimulation, and Regulation of Cellular Activation*

*Effect of cotreatment with agonist of OPRM1 (DAMGO) and agonist of CD-14 receptor (LPS) on NF-κΒ*

U50488 14.151,3 P < 0.00011 14.154,6 p < 0.0014

p < 0.0001

,

**Paired T-test Cell population in S** 

**phase (%)**

**Paired T-test**

p = 0.00026

8.075,6 p < 0.0001<sup>5</sup>

Our studies show that U50488, agonist for κOR, was able to mitigate the effect of DAMGO, an agonist for MOR and a significant reduction in the percentage of cells in both M-phase and S-phase of cell cycle was observed at 2 and 24 h of

Receptor-receptor interactions and di/oligomerization are established pathways for altering response to any given ligand. As both homo- and heterodimers of μOR have been reported, it is speculated that dimerization of μOR may have a role in regulating receptor signaling. This intermolecular cross talk within receptor oligomers often results in allosterism between the different binding pockets of the individual monomers. Negative binding cooperativity has been observed for both GPCR homo- and heteromers using equilibrium binding and/or radio ligand

Assays with TLR and μOR coactivation showed that agonist activation of μOR using DAMGO causes a significant lowering of LPS-induced cell division in cells coexpressing μOR and TLR. This has direct bearing on pathogen clearance as TLRs are present and expressed in sentinel cells such as macrophages and dendritic cells, which recognize structurally conserved molecules derived from microbes. These large phagocytes are found in essentially all tissues where they patrol for potential pathogens by amoeboid movement. These cells play a critical role in nonspecific

**7. Effect of μOR coactivation on cell proliferation**

*Effect of U50488 and DAMGO on cell proliferation at 2 h after treatment.*

*DOI: http://dx.doi.org/10.5772/intechopen.81568*

**Treatment Cell population in** 

**M phase (%)**

DAMGO 26.661,2 9.404,5

17.862,3 P < 0.0001<sup>2</sup>

*Immune Cell Activation: Stimulation, Costimulation, and Regulation of Cellular Activation DOI: http://dx.doi.org/10.5772/intechopen.81568*

#### **Figure 3.**

*Immune Response Activation and Immunomodulation*

**Table 3** and **Figure 3**.

**NF-**κΒ **phosphorylation**

**Treatment Cell population in** 

**M phase (%)**

LPS + DAMGO 3.892,3 P < 0.0001<sup>2</sup>

*Effect of LPS and DAMGO on cell proliferation at 2 h after treatment.*

LPS 6.071,2 15.74, 5

phagocytic receptors that stimulate ingestion of the pathogens they recognize. Some are chemotactic receptors, such as the f-Met-Leu-Phe receptor, which binds the N-formylated peptides produced by bacteria and guide neutrophils to sites of infection. A third function, which may be mediated by some of the phagocytic receptors as well as by specialized signaling receptors, is to induce effector molecules that contribute to induce innate immune responses and molecules that influence the initiation and nature of any subsequent adaptive immune response [1]. Various immune cell functions regulated by receptors on immunocytes are summarized in

**5. CD14 receptor coactivation with μOR: effect on cell division and** 

age of cells in M-phase as well as in S-phase of the cell cycle (**Tables 2** and **3**).

DAMGO 4.831,3 P < 0.00011 10.54, 6 p = 0.0294

p = 0.00183

,

**Paired T-test Cell population in** 

**S-phase (%)**

**Paired T-test**

p = 0.0026

,

7.995, 6 p = 0.0001<sup>5</sup>

**6. Effect of μOR activation with a selective ΚOR agonist and μOR** 

The effect of coactivation of two different opioid receptors, U50488, a selective KOR agonist was used for lymphocyte activation, and effect of costimulation of μOR was achieved by using DAMGO, an opioid receptor agonist. Cells were treated with μOR agonists DAMGO for varying time intervals, and effect on cell proliferation and NF-κΒ phosphorylation was studied. Results using HepG2 revealed that U50488 (an agonist for KOR) was able to mitigate the effect of DAMGO, an agonist for μOR, and a significant reduction in the percentage of cells in both M-phase and S-phase of the cell cycle was observed at 2 and 24 h of treatment. U50488 alone also showed a larger percentage of cells in G0 phase both at 2 and 24 h in comparison with DAMGO, and the mitigation of U50488 induced effects indicating cross talk of receptors (**Figure 3**).

LPS-mediated lymphocyte activation and effect of costimulation with opioid receptor agonists were studied by treating the cells with the μOR agonist DAMGO for time intervals of 5, 30, or 240 min. A549, a cell line having both CD14 and μOR, was used to study the effect on cell proliferation and NFκΒ phosphorylation. Treatment of A549 revealed that DAMGO was able to mitigate the LPS-mediated induction of phosphorylated NFκB after cotreatment for 4 h (**Figure 2**). Similarly, DAMGO was also able to suppress the cell proliferation by LPS, significantly reducing the percent-

**90**

*1 NT vs. LPS.*

*2*

*3*

*4*

*5*

*6*

**Table 3.**

*NT vs. DAMGO.*

*LPS vs. DAMGO.*

*NT vs. LPS + DAMGO.*

*LPS vs. LPS + DAMGO.*

*DAMGO vs. LPS + DAMGO.*

**antagonist**

*Effect of cotreatment with agonist of OPRM1 (DAMGO) and agonist of CD-14 receptor (LPS) on NF-κΒ phosphorylation.*


#### **Table 4.**

*Effect of U50488 and DAMGO on cell proliferation at 2 h after treatment.*

Our studies show that U50488, agonist for κOR, was able to mitigate the effect of DAMGO, an agonist for MOR and a significant reduction in the percentage of cells in both M-phase and S-phase of cell cycle was observed at 2 and 24 h of treatment (**Table 4**).
