**3. Opioid receptor and immune function**

As early as 1987, Jankovic and Maric [4] showed that the neuropeptides methionine-enkephalin, and leucine-enkephalin, exhibit a protective action against anaphylactic shock in rats sensitized to ovalbumin. Subsequent studies have shown that enkephalins can act both as suppressors and potentiators of immune response in a dose-dependent manner. Animal studies, where nutritional status, environmental influences, history of drug abuse, and genetic variability can be controlled more easily, have shown that morphine treatment results in significant immune deficits. Chronic morphine use has been shown to result in severe immunosuppression, posing as a significant risk factor for opportunistic infection [5], and this finding is also supported by epidemiological studies that show an increased prevalence of opportunistic infections in opiate users [6]. Chronic morphine has been shown to effect early reactions of innate immunity and later responses of adaptive immunity against microbes [7]. In addition, morphine has also been shown to affect the brainimmune axis by an IL-1β-dependent pathway [8]. Various studies support the idea that chronic morphine exposure in vivo attenuates lymphocyte proliferation [9], NK cell cytotoxicity [10], antibody and serum hemolysin formation [11], and phagocytic properties of peripheral mononuclear leukocytes [12]. Morphine exposure has also been shown to increase mortality of infected mice [12–14]. Novick et al. [15] showed that long-term abuse of opiates results in impaired NK cell activity and altered CD4+

*Immune Response Activation and Immunomodulation*

**2. Mechanism of activation of adapted immunity**

• cell-mediated immune responses carried out by T cells

• antibody responses carried out by B cells

cytic cells of the innate immune system [3].

intervention (**Table 1**).

classes:

on a time scale of minutes to hours. Several cell types in this system are capable of activation to secrete cytokines, rapidly proliferate, or otherwise communicate to surrounding cells that there is a pathogen to consider. Upon clearance of the pathogen, the cell population must contract in a controlled manner. Furthermore, in some cell populations (e.g., T cells), a subset of cells is retained as long-lived memory cells to protect and prime the system for future insults. Researchers are increasingly focused on early events in immune cell activation, where the response to an inflammatory signal can be tuned to impact overall cell function. In areas such as immuno-oncology, increased activation is connected to improve cell expansion whereas in the field of immunosuppression, the converse is desired. Clinical successes in targeting the immune system for treating cancer have generated a resurgence of effort to harness the immune system more routinely for therapeutic

Adaptive immune responses carried out by lymphocytes are of two broad

During an immune response, the B cells are activated to secrete immunoglobulins, which circulate in blood, permeate to other body fluids, and bind specifically to foreign antigen that stimulated their production in the first place. This binding inactivates viruses and microbial toxins by blocking their interaction with the host cells. Antibody binding also marks invading pathogens for destruction by phago-

Cell-mediated reactions depend on direct interactions between T lymphocytes and cells bearing the antigen that the T cells recognize. T cells are specialized to recognize foreign antigens as peptide fragments bound to proteins of the major histocompatibility complex (MHC). The cytotoxic T cells recognize any infected cells with the help of viral antigens displayed on the surface of the infected cells [1]. Other T lymphocytes that activate the cells they recognize are marked by the expression of the cell-surface molecule CD4 on helper T cells. The CD4 T lymphocytes can be divided into two subsets, which carry out different functions by defending the body particularly from bacterial infections. Bacteria phagocytosed by macrophages are destroyed in the lysosomes, which contain several enzymes and antimicrobial substances. The intracellular bacteria, as in case of tuberculosis (Mtb), survive, because the vesicles they occupy do not fuse with the lysosomes. These infections are modified by a subset of CD4 T cells, namely TH1 cells, which activate macrophages, induce fusion of lysosomes and phagocytic vesicles containing the bacteria, and at the same time stimulate other antibacterial mechanisms of the phagocyte. CD4+ T cells play critical role during Mtb infection by mediating protection, contributing to inflammation, and regulating immune response. Th1 and Th17 cells are the main effector CD4+ T cells during Mtb. Th1 cells release cytokines and chemokines that attract phagocytes to the site of infection and impart protection from Mtb by secreting IFN-γ and activating antimycobacterial action in

**82**

macrophages.

and CD8+ T cell numbers. In animal models, parenteral use of opiates was shown to inhibit mitogenic and effector cell responses in both B and T cells [9, 16].

### **3.1 Opioid receptors in various immune cell types and their functional implications**

Molecular biology studies have shown that immune cells differentially express opioid receptors (OR), and morphine affects their development, differentiation, and function [17]. Binding sites and protein expression for delta (δ) and kappa (κ) subclasses of G protein-coupled ORs [18, 19], in addition to gene expression of δ, κ, and μ subclasses [20, 21], have been described in leukocytes. Chuang et al. [20] reported the presence of mRNA for the μOR in human T- and B-cell lines, CD4+ T cells, monocytes, macrophages and granulocytes. Retinoid receptor activation increases the expression of the μOR in U937 cells, a mononuclear cell line [22]. Mu (μ-), Kappa (κ-), and Delta (δ-) opioids have been shown to possess chemoattractant activity and induce the chemotaxis of both monocytes and neutrophils [23–28]. Simpkins et al. [24] and Van Epps and Saland [25] showed that opiates, acting through δ and μ subclasses of OR expressed on human monocytes and neutrophils, are capable of inhibiting subsequent migratory responses to chemokines, and that this process of heterologous desensitization/trans-deactivation is associated with phosphorylation of chemokine receptors. Grimm et al. [26] showed that phagocytes respond chemotactically, with a chemotaxis index 2- to 2.5-fold higher than controls, to met-enkephalin and morphine, and this chemotaxis was inhibited by the OR antagonist naloxone. Liu et al. [27] demonstrated that pretreatment with opioids, including morphine, heroin, met-enkephalin, the selective μ-agonist DAMGO, or the selective δ-agonist [D-Pen2, D-Pen5] enkephalin (DPDPE), leads to the inhibition of the chemotactic response of leukocytes to complement-derived chemotactic factors. They also affect the chemokines macrophage inflammatory protein (MIP-1α)/CCL3, RANTES/CCL5, monocyte chemotactic protein-1 (MCP-1)/CCL2, and IL-8/CXCL8 [28]. Many investigators choose to study the effects of morphine on immune function because morphine has clinical applications and shows good affinity for all three types of ORs. However, use of morphine as the opioid of choice has limited the ability to delineate, which type of OR mediates the given immunological response/s due to binding to all receptor types.

Existence of a low-affinity, naloxone-insensitive morphine binding site on human peripheral blood macrophages has also been reported [29]. Opioid alkaloids, such as morphine and the endogenous peptides, including β-endorphin and dynorphin, directly modulate the function of lymphocytes and other cells involved in host defense and immunity, ORs preferentially bind to the (−)-enantiomer of most opioid alkaloids, for example, ORs will bind the antagonist (−)-naloxone but not (+)-naloxone [30].

#### **3.2 Immunosuppression mediated by opiates**

The role of opiate drugs in suppressing a variety of immunological endpoints such as proliferation, functions and responses of both T and B cells, and attenuation of the cytokine system has been studied extensively [31, 32]. Opiate drug administration has also been reported to suppress movement and number of white blood cells [33, 34]. Heroin use has been documented to depress E-rosette formation indicating clinical immune suppression [35]. Long-term use of opiate drugs has been reported to depress T cell reactivity and cause a loss of T helper (TH) cells [36, 37], reduces T helper/T cytotoxic cell ratios, and decreases T helper cell function [38–41]. Use of opiate drugs produces atrophy of lymphoid organs, decreases lymphoid content, and alters antigen-specific antibody production [42, 43]. Opiate

**85**

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

addiction induces immunonutritional deficiencies [44, 45] and impairs immunoglobulin synthesis and function [46]. Naik et al. [47] showed a decrease of IgA levels and increase of IgG levels in Indian opiate users as compared to nonusers. Opioids bind directly on immune cells and modulate the function of these cells and also bind to classical ORs in the CNS, causing the release of catecholamines and/or steroids, which in turn also affect the immune cells. At the same time, morphine is known to activate the hypothalamic-pituitary-adrenal axis and release glucocorticoids, which

**3.3 Impact of functional polymorphism in OPRM1 gene on cell function**

These results suggest that cellular environment may influence the phenotype associated with the variant receptor. Deb et al*.* [53], using murine neuroblastoma Neuro 2 A cells stably transfected with cDNA containing 118G variant, studied the effect on PKA, ERK, and CREB activation and documented no upregulation of PKA activity but a differential response of ERK phosphorylation in comparison to 118A variant, following chronic morphine treatment. Zhang et al*.* [54] analyzed 87 human brain tissue samples derived from autopsies and performed in vitro experiments on Chinese hamster ovary (CHO) cells, to show that the amount of mRNA transcribed from the 118G allele was twofold lower than the mRNA derived from the 118A allele. The levels of variant protein were ten-fold lower compared with those of the wild-type receptor. They also showed that after transfection into CHO cells with a cDNA representing only the coding region of *OPRM1* and inhibition of transcription with actinomycin D, the mRNA turnover was same for 118A and 118G variants. An *in silico* study by Pang et al*.* [55] showed that the substitution of the A with G at position 118 of the *OPRM1* gene abolishes three transcription factor binding sites, while creating a novel exon splice enhancer as well as p53 and a zinc finger protein binding sites, predicting a direct effect of 118A > G on gene expression and on the processing of heterogeneous nuclear RNA into mature mRNA. Huang et al*.* [56] described the role of the 118A > G SNP in posttranslational mechanisms suggesting that N-glycosylation may affect receptor expression, since it plays an important role in correct folding of receptors in the endoplasmic reticulum and, hence, their sorting from the endoplasmic reticulum to the plasma membrane. Huang et al*.* [56] also showed that the variant receptor had lower relative molecular mass than the wild-type one, which may be explained by a differential glycosylation status between the two receptors. Pulse-chain (or chase) experiments revealed that the

Several studies suggest that immune cells contain μORs along with existence of morphine binding sites differing from classical μORs, and measurements of the mRNAs that encode the neuronal types of OR show low levels of receptor mRNA in immune cells [49]. μOR is known to depict a total of 43 variants within coding and noncoding regions of the *OPRM1* gene, and 52 different haplotypes were predicted in the subgroup of African Americans. These haplotypes were classified by similarity clustering into functionally related categories, and one of these was significantly more frequent in substance-dependent individuals, viz. [−1793T-A, −1699insT, −1320A-G, −111C-T, +17C-T (+118A-G)], which was associated with substance dependence [50]. Studies evaluating the effects of 118A > G SNP on the intracellular signaling cascades resulting from μ-OR activation have shown conflicting results. Both DAMGO and morphine were twofold more potent in inhibiting calcium channel currents in sympathetic neurons transfected with the 118G allele than in neurons expressing the wild-type receptors [51]. However, Kroslak et al. [52] showed in HEK293 and AV-12 cells that stable expression of the 118G variant was associated with decreased agonist-mediated cyclic adenosine monophosphate (cAMP) signaling for morphine, methadone, and DAMGO, but not for β-endorphin.

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

are immunosuppressive in their own capacity [48].

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

addiction induces immunonutritional deficiencies [44, 45] and impairs immunoglobulin synthesis and function [46]. Naik et al. [47] showed a decrease of IgA levels and increase of IgG levels in Indian opiate users as compared to nonusers. Opioids bind directly on immune cells and modulate the function of these cells and also bind to classical ORs in the CNS, causing the release of catecholamines and/or steroids, which in turn also affect the immune cells. At the same time, morphine is known to activate the hypothalamic-pituitary-adrenal axis and release glucocorticoids, which are immunosuppressive in their own capacity [48].

### **3.3 Impact of functional polymorphism in OPRM1 gene on cell function**

Several studies suggest that immune cells contain μORs along with existence of morphine binding sites differing from classical μORs, and measurements of the mRNAs that encode the neuronal types of OR show low levels of receptor mRNA in immune cells [49]. μOR is known to depict a total of 43 variants within coding and noncoding regions of the *OPRM1* gene, and 52 different haplotypes were predicted in the subgroup of African Americans. These haplotypes were classified by similarity clustering into functionally related categories, and one of these was significantly more frequent in substance-dependent individuals, viz. [−1793T-A, −1699insT, −1320A-G, −111C-T, +17C-T (+118A-G)], which was associated with substance dependence [50]. Studies evaluating the effects of 118A > G SNP on the intracellular signaling cascades resulting from μ-OR activation have shown conflicting results. Both DAMGO and morphine were twofold more potent in inhibiting calcium channel currents in sympathetic neurons transfected with the 118G allele than in neurons expressing the wild-type receptors [51]. However, Kroslak et al. [52] showed in HEK293 and AV-12 cells that stable expression of the 118G variant was associated with decreased agonist-mediated cyclic adenosine monophosphate (cAMP) signaling for morphine, methadone, and DAMGO, but not for β-endorphin. These results suggest that cellular environment may influence the phenotype associated with the variant receptor. Deb et al*.* [53], using murine neuroblastoma Neuro 2 A cells stably transfected with cDNA containing 118G variant, studied the effect on PKA, ERK, and CREB activation and documented no upregulation of PKA activity but a differential response of ERK phosphorylation in comparison to 118A variant, following chronic morphine treatment. Zhang et al*.* [54] analyzed 87 human brain tissue samples derived from autopsies and performed in vitro experiments on Chinese hamster ovary (CHO) cells, to show that the amount of mRNA transcribed from the 118G allele was twofold lower than the mRNA derived from the 118A allele. The levels of variant protein were ten-fold lower compared with those of the wild-type receptor. They also showed that after transfection into CHO cells with a cDNA representing only the coding region of *OPRM1* and inhibition of transcription with actinomycin D, the mRNA turnover was same for 118A and 118G variants. An *in silico* study by Pang et al*.* [55] showed that the substitution of the A with G at position 118 of the *OPRM1* gene abolishes three transcription factor binding sites, while creating a novel exon splice enhancer as well as p53 and a zinc finger protein binding sites, predicting a direct effect of 118A > G on gene expression and on the processing of heterogeneous nuclear RNA into mature mRNA. Huang et al*.* [56] described the role of the 118A > G SNP in posttranslational mechanisms suggesting that N-glycosylation may affect receptor expression, since it plays an important role in correct folding of receptors in the endoplasmic reticulum and, hence, their sorting from the endoplasmic reticulum to the plasma membrane. Huang et al*.* [56] also showed that the variant receptor had lower relative molecular mass than the wild-type one, which may be explained by a differential glycosylation status between the two receptors. Pulse-chain (or chase) experiments revealed that the

*Immune Response Activation and Immunomodulation*

**implications**

and CD8+ T cell numbers. In animal models, parenteral use of opiates was shown to

Molecular biology studies have shown that immune cells differentially express opioid receptors (OR), and morphine affects their development, differentiation, and function [17]. Binding sites and protein expression for delta (δ) and kappa (κ) subclasses of G protein-coupled ORs [18, 19], in addition to gene expression of δ, κ, and μ subclasses [20, 21], have been described in leukocytes. Chuang et al. [20] reported the presence of mRNA for the μOR in human T- and B-cell lines, CD4+ T cells, monocytes, macrophages and granulocytes. Retinoid receptor activation increases the expression of the μOR in U937 cells, a mononuclear cell line [22]. Mu (μ-), Kappa (κ-), and Delta (δ-) opioids have been shown to possess chemoattractant activity and induce the chemotaxis of both monocytes and neutrophils [23–28]. Simpkins et al. [24] and Van Epps and Saland [25] showed that opiates, acting through δ and μ subclasses of OR expressed on human monocytes and neutrophils, are capable of inhibiting subsequent migratory responses to chemokines, and that this process of heterologous desensitization/trans-deactivation is associated with phosphorylation of chemokine receptors. Grimm et al. [26] showed that phagocytes respond chemotactically, with a chemotaxis index 2- to 2.5-fold higher than controls, to met-enkephalin and morphine, and this chemotaxis was inhibited by the OR antagonist naloxone. Liu et al. [27] demonstrated that pretreatment with opioids, including morphine, heroin, met-enkephalin, the selective μ-agonist DAMGO, or the selective δ-agonist [D-Pen2, D-Pen5] enkephalin (DPDPE), leads to the inhibition of the chemotactic response of leukocytes to complement-derived chemotactic factors. They also affect the chemokines macrophage inflammatory protein (MIP-1α)/CCL3, RANTES/CCL5, monocyte chemotactic protein-1 (MCP-1)/CCL2, and IL-8/CXCL8 [28]. Many investigators choose to study the effects of morphine on immune function because morphine has clinical applications and shows good affinity for all three types of ORs. However, use of morphine as the opioid of choice has limited the ability to delineate, which type of OR mediates the

inhibit mitogenic and effector cell responses in both B and T cells [9, 16].

given immunological response/s due to binding to all receptor types.

**3.2 Immunosuppression mediated by opiates**

Existence of a low-affinity, naloxone-insensitive morphine binding site on human peripheral blood macrophages has also been reported [29]. Opioid alkaloids, such as morphine and the endogenous peptides, including β-endorphin and dynorphin, directly modulate the function of lymphocytes and other cells involved in host defense and immunity, ORs preferentially bind to the (−)-enantiomer of most opioid alkaloids, for example, ORs will bind the antagonist (−)-naloxone but not (+)-naloxone [30].

The role of opiate drugs in suppressing a variety of immunological endpoints such as proliferation, functions and responses of both T and B cells, and attenuation of the cytokine system has been studied extensively [31, 32]. Opiate drug administration has also been reported to suppress movement and number of white blood cells [33, 34]. Heroin use has been documented to depress E-rosette formation indicating clinical immune suppression [35]. Long-term use of opiate drugs has been reported to depress T cell reactivity and cause a loss of T helper (TH) cells [36, 37], reduces T helper/T cytotoxic cell ratios, and decreases T helper cell function [38–41]. Use of opiate drugs produces atrophy of lymphoid organs, decreases lymphoid content, and alters antigen-specific antibody production [42, 43]. Opiate

**3.1 Opioid receptors in various immune cell types and their functional** 

**84**

half-life of the mature form of the variant receptor (~12 h) was shorter than that of the wild-type receptor (~28 h) showing its effect on protein stability. Thus, several lines of evidence suggest that the 118G variant may affect *OPRM1* gene expression in addition to mRNA translation, posttranslational processing, or turnover of the μ-opioid receptor protein, which can all effect signaling pathway/s.
