Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Endogenous opioid peptides are well known for mediating analgesic activity at the µ-, -, and -opioid receptors, which are widely distributed in the nervous system. Over the past several years, opioid peptides and alkaloids have come to be accepted as modulators of the immune system as well. A wealth of accumulated evidence has shown opioid-induced regulation of various immune cell responses such as lymphocyte proliferation (Kowalski, 1998), antibody production (Taub et al., 1991), macrophage (M) phagocytosis (Ichinose et al., 1995), and M tumoricidal activity (Foster and Moore, 1987). Immunoenhancing or immunosuppressive responses have been characterized and were dependent on the concentration and class of opioid (µ, , or ), as well as on the type and status of immune cell studied. In addition to their capacity to regulate immune cell functions, opioid peptides also are produced by various populations of immune cells (Smith and Blalock, 1981). These findings provide strong evidence for autocrine and paracrine modes of communication directly linking the immune and nervous systems. Detection of mRNA encoding for µ (Sedqi et al., 1995),  (Chuang et al., 1994; Gaveriaux et al., 1995), and  (Belkowski et al., 1995; Gaveriaux et al., 1995) opioid receptors in leukocytes suggests further the existence of these neuronal receptors on immune cell populations, lending further credence to the participation of opioids in neural-immune signaling networks.

Binding studies characterizing brain-like -opioid receptors on the R1.1 thymoma cell line also suggest the possibility of these neuronal receptors on immune cells (Bidlack et al., 1992; Lawrence and Bidlack, 1992). However, high-affinity, stereoselective binding that was inhibited by selective opioids has been difficult to detect on primary, heterogeneous immune cell populations using radioligand-binding techniques (Sibinga and Goldstein, 1988). This lack of consistent, reproducible pharmacological evidence had prompted a search for a more sensitive means to detect and characterize opioid receptors on these cells. Recently, our laboratory has reported an alternative approach for the identification of receptors through the use of an indirect, immunofluorescent-labeling procedure (Lawrence et al., 1995a, 1997; Ignatowski and Bidlack, 1998). Successful labeling of the -opioid receptor on a derivative of R1.1 thymoma cells, the R1EGO (R1E/TL8x.1.G1.OUA^r.1) thymoma cell line (Lawrence et al., 1995a, 1997; Ignatowski and Bidlack, 1998), which is rich in -opioid receptor expression (Lawrence et al., 1995b), as well as on freshly isolated mouse thymocytes (Lawrence et al., 1995a; Ignatowski and Bidlack, 1998), has been attained. This sensitive method employs amplification of labeling by a -selective opioid, fluorescein-conjugated 2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3-aminophenyl)-2-(1-pyrrolidinyl)ethyl] acetamide (FITC-AA) (Lawrence et al., 1995a) with the addition of biotinylated antifluorescein IgG and extravidin-R-phycoerythrin (PE). In the absence of this amplification procedure, specific opioid labeling was undetectable for either R1EGO cells or thymocytes using fluorophore-conjugated opioid ligands (Lawrence et al., 1997).

Thymocytes, consisting mainly of immature T cells, demonstrate specific -opioid receptor labeling (Ignatowski and Bidlack, 1998) as assessed in the presence of the -selective antagonist nor-binaltorphimine (nor-BNI; Portoghese et al., 1987). Neither µ- nor -selective opioids were able to effectively displace the FITC-AA/PE labeling, further demonstrating the specificity of labeling for the -opioid receptor (Ignatowski and Bidlack, 1998). The question remained, however, as to whether mature immune cells express the -opioid receptor. To address this issue of -opioid receptor expression on mature immune cells, splenocytes (a source of mature T and B cells), as well as resident, peritoneal M (a source of mature M) and thioglycollate (TG)-elicited M (used to enrich for M) were labeled for the -opioid receptor using this amplification procedure. Simultaneous labeling of cells with fluorophore-conjugated monoclonal antibodies (mAbs) directed against specific CD cell surface markers provided for the identification of subpopulations of immune cells within larger, heterogeneous populations. In the present report, data are presented regarding the differential expression of the -opioid receptor on various immune cell populations at varying stages of differentiation or phenotypic maturation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Mice. Male C57BL/6ByJ mice, aged 6 to 8 weeks (Jackson Laboratories, Bar Harbor, ME), were used for all studies. The mice were given access to food and water ad libitum.

Murine Splenocyte Preparation. Mice were sacrificed by CO₂ inhalation, and spleens were removed aseptically as described previously (Bidlack et al., 1996). Splenocytes were dissociated by gently pressing the spleens between the frosted ends of sterile microscope slides in ice-cold, HEPES-buffered balanced salt solution (HEPES-BSS), consisting of 15 mM HEPES, 3.4 mM K₂HPO₄, 0.6 mM KH₂PO₄, 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, and 1% BSA, pH 7.4. Cell suspensions were passed over sterile, glass-wool columns to remove dead cells and debris. After centrifugation at 200g for 10 min at 4°C, cell suspensions were treated with a cold, isotonic ammonium chloride solution to lyse contaminating erythrocytes. Unfixed cells, washed twice by centrifugation at 200g for 10 min at 4°C and counted in a Coulter Z1 Counter, were resuspended at a final concentration of 2 × 10⁶ cells in 200 µl HEPES-BSS for optimal staining of the -opioid receptor on B cells as described below. Enrichment of mature, splenic T cells was achieved by further passage of the cell suspension over a nylon-wool column eluted with 37°C HEPES-BSS. After centrifugation at 200g for 10 min at 4°C, fluorescent opioid labeling of the cells was measured as described below.

Peritoneal M. Thioglycollate (sterile, 3% solution; aged 2-4 months before use; Sigma Chemical Co., St. Louis, MO) was injected i.p. (1.5 ml/mouse) to invoke the production of an inflammatory exudate rich in M. Elicited M were harvested and pooled from 4 to 5 mice, and resident peritoneal M were harvested and pooled from 10 to 15 mice by peritoneal lavage. Ice-cold PBS (without Ca²⁺ and Mg²⁺) was used for all M harvests, as well as in the -opioid receptor-labeling procedure for M. All M samples, in addition to 1% BSA, contained 10% rabbit (Rb) serum to decrease further nonspecific staining in the labeling procedure, which is similar to that described below for lymphocytes. For in vivo M activation experiments, each mouse was injected i.p. with 10 µg of lipopolysaccharide (LPS) (20 µg/ml; Escherichia coli type 0111:B4; Sigma Chemical Co.) or with 0.5 ml of sterile, 0.9% sodium chloride solution, the LPS vehicle control. Various times after saline or LPS injection, mice were sacrificed and M were harvested and pooled from 10 mice/group. Resident, peritoneal M were used as an additional control group for the M activation studies because, as noted by others (Fisker et al., 1992), the i.p. injection of sterile saline induced a mild inflammatory reaction as revealed by an increased polymorphonuclear cell immigration into the peritoneum.

Indirect Immunofluorescent and Phenotypic Labeling. The FITC-AA labeling and amplification procedure were performed as described previously (Lawrence et al., 1995a; Ignatowski and Bidlack, 1998). In a final volume of 200 µl HEPES-BSS, cells were incubated with 30 µM FITC-AA for 30 min at 25°C for optimal staining. The -selective antagonist nor-BNI (500 µM) was included to measure nonspecific fluorescence. Samples were chilled on ice, diluted with 1 ml of HEPES-BSS, and centrifuged at 400g for 3 min at 4°C. After aspirating the supernatants, cells were washed twice and resuspended in a final volume of 100 µl of HEPES-BSS, containing 10 µl of biotinylated Rb antifluorescein IgG, except in FITC-AA- and PE-only controls, in which the Rb Ab was omitted. After incubation for 30 min at 4°C in the dark, samples were diluted with 1 ml of HEPES-BSS and centrifuged at 400g for 3 min at 4°C. The supernatants were aspirated, and the cells were washed again. Cells were resuspended in 40 µl of HEPES-BSS and 10 µl of PE for 15 min at 4°C in the dark. The cells were washed twice as described above and were resuspended in a final volume of 1 ml of HEPES-BSS for flow cytometric analysis.

Flow Cytometric Analysis. Samples were analyzed on a Becton-Dickinson FACScan flow cytometer (San Jose, CA) equipped with a 15-mW argon-ion laser for excitation (488 nm) of FITC and PE using band-pass filters of 530 ± 15 nm and 585 ± 21 nm, respectively. The red fluorescence channel (FL3) for QR (which emits at 670 nm) uses a long-pass emission filter that transmits long-wavelength light beams above 650 nm. In each sample, 25,000 lymphocytes or M were analyzed.

Data Analysis. Data [forward angle and right angle (side) light scatter, as well as green (FITC), orange (PE), and red (QR) fluorescence] were measured, collected, and stored into list-mode data files using a Macintosh Quadra 650 computer system with CELLQuest software (Becton-Dickinson Immunocytometry Systems). Median peak values of relative fluorescence intensity distributions were used to compare the fluorescence among samples, which assumed that only a single population was labeled. Viabilities were >95% as assessed by propidium iodide (Sigma Chemical Co.) exclusion. To report a relative quantification for -opioid receptor labeling of subpopulations of immune cells identified by mAb labeling, gating and histogram subtraction were performed using CELLQuest software as described previously (Ignatowski and Bidlack, 1998).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Phenotypic Characterization of Splenocytes as Assessed by Flow Cytometry. To determine whether preferential -opioid receptor expression exists on splenocyte subpopulations, it was first necessary to phenotypically characterize the mouse immune cells of interest. Splenocytes, as compared with thymocytes, differ vastly in the phenotypic profile of their lymphocyte populations (Table 1). Various FITC-, PE-, and QR-conjugated mAbs directed against mouse cell surface markers were used for phenotypic-labeling experiments to define the immune cells analyzed. As reported previously (Ignatowski and Bidlack, 1998), simultaneous labeling of thymocytes with the T-helper cell marker, CD4, and with the mAb used to identify T-cytotoxic cells, CD8, demonstrated that the majority of thymocytes labeled with both mAbs, identifying them as double-positive, immature T cells. Few mature T-helper or T-cytotoxic cells were located within the thymus. In contrast, the spleen virtually lacked double-positive, immature T cells (0.35 ± 0.1%). In single-labeling experiments, it was determined that approximately 40% of the splenocytes were T cells, either T-helper cells (CD4⁺) or T-cytotoxic cells (CD8⁺; Table 1), which was confirmed by double-labeling experiments (CD4⁺/CD8, 23 ± 2%; CD8⁺/CD4, 16 ± 2%). Using the pan B cell marker CD45R, the majority of remaining cells were identified as B cells, of which 76% were shown to be mature B cells by labeling with the IgD^b mAb (Table 1). The remaining cells within the spleen were identified as either natural killer (NK) cells or M based on labeling using their respective phenotypic mAb markers (Table 1). These phenotypic splenocyte results are similar to those reported by Beavis and Pennline (1994).

View this table: [in this window] [in a new window]   TABLE 1 Lymphocyte percentages based on phenotypic analysis Gated thymocytes (Ignatowski and Bidlack, 1998) or splenocytes were stained with mAbs directed against either CD8, CD4, CD45R, IgDb, NK1.1, or Mac-3. Percentages of positive cells were computed by subtracting control isotype histograms (QR or FITC background) from mAb-stained samples. Data are mean ± S.E. of three or four separate experiments, each performed in triplicate.

-Opioid Receptor Labeling of Mouse Splenocytes. The extent of -opioid receptor expression on the phenotypically defined splenocyte subpopulations next was determined by using the amplification procedure for -opioid receptor labeling along with staining of cells by QR-conjugated mAbs to identify the particular cells of interest. Analysis of double-labeled splenocytes to identify whether T-helper cells (CD4⁺), T-cytotoxic cells (CD8⁺), or B cells (CD45R⁺) expressed the -opioid receptor was undertaken as described previously (Ignatowski and Bidlack, 1998). For example, in the histogram of QR-CD4 fluorescently labeled splenocytes (Fig. 1A), a region was set to enclose the CD4⁺ cells. This region then was selected and analyzed in the PE fluorescence histogram for determination of -opioid receptor expression (Figs. 1B and 2A). Subtraction of background PE median peak fluorescence intensity demonstrated a reduction in the median fluorescence intensity values with FITC-AA/PE when nor-BNI was included as an inhibitor (Fig. 2B and Table 2). The percentage of specific labeling for the -opioid receptor was approximately the same regardless of subpopulation analyzed (Table 2), suggesting that one population of -opioid receptor was expressed on multiple lymphocyte subsets.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Flow cytometric analysis of CD4+ splenocytes labeled for the -opioid receptor. Splenocytes were labeled via the amplification procedure for the -opioid receptor and/or anti-CD4-QR. A, CD4+ splenocytes were gated (R2) in the FL3 (QR) fluorescence histogram. These gated cells then were viewed in the FL2 (PE) fluorescence channel for subsequent -opioid receptor-labeling analysis. B, total labeling of CD4+ splenocytes for the -opioid receptor is demonstrated in relation to the autofluorescence control for the whole splenocyte population. In the absence of added PE, the arbitrary fluorescence units were 0.3.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   -opioid receptor labeling of CD4+ splenic lymphocytes. CD4+ splenocytes were labeled via the amplification procedure for the -opioid receptor. Control samples were labeled with an isotype-matched (IgG2a) control mAb conjugated to QR. A, -opioid receptor labeling of CD4+ splenocytes is demonstrated by overlaid histograms. B, specific labeling of the -opioid receptor on CD4+ splenocytes as demonstrated after histogram subtraction of the background PE fluorescence. Note the downward and leftward shift of the PE histogram in the presence of nor-BNI. Data represent four separate experiments.

View this table: [in this window] [in a new window]   TABLE 2 Phenotype-specific splenocyte subsets labeled for the -opioid receptor Double-labeling of splenocytes for both the -opioid receptor and CD4, CD8, or CD45R was performed as described in Materials and Methods and as published previously (Bidlack et al., 1996; Ignatowski and Bidlack, 1998). Data are the mean ± S.E. of number of experiments in parentheses performed in triplicate.

Differential -Opioid Receptor Expression on Immature Lymphocytes Compared with Mature Lymphocytes. Additional analysis of the double-labeled splenocyte subpopulations provided further insight into the distribution of the -opioid receptor on mouse immune cells. CELLQuest software was used to perform histogram (FL2 fluorescence channel for PE) subtraction of cells labeled for each mAb and the -opioid receptor in the presence of the -selective opioid nor-BNI (nonspecific labeling) from those cells labeled with each mAb and the -opioid receptor in the absence of nor-BNI (total labeling) on a channel-by-channel basis. This subtraction procedure yielded the resultant relative percentage of cells specifically labeled for both the mAb of interest and the -opioid receptor. These results are presented in Table 2. Previously, it was shown that whether thymocytes were identified using CD8 or CD4 mAbs, the relative percentage of cells expressing the -opioid receptor was similarapproximately 60% (Ignatowski and Bidlack, 1998). This finding was not surprising because the majority of all thymocytes were identified as CD4⁺/CD8⁺ immature T cells (Ignatowski and Bidlack, 1998). However, whereas B cells and mature T cells from the spleen also demonstrated specific labeling for the -opioid receptor, a much smaller percentage of cells within these subpopulations expressed the -opioid receptor. Less than 25% of the mature T cells and only 16% of B cells labeled for the -opioid receptor (Table 2).

-Opioid Receptor Labeling of Resident and TG-Elicited Peritoneal M. M, another immunologically, well characterized cell population, were analyzed for -opioid receptor expression. Gating on the whole PEC population, which consisted of M, lymphocytes, and polymorphonuclear cells (Eichner and Smeaton, 1983; Melnicoff et al., 1989), and resident and TG-elicited M were identified for analysis in flow cytometry based on their distinctive forward scatter (FSC) versus side scatter (SSC) cellular characteristics (Ho and Springer, 1983; Hendrzak et al., 1994; Plasman and Vray, 1994) as well as on their labeling with the Mac-3 mAb, which was used for the detection of resident and inflammatory-elicited M, but not monocytes, lymphocytes, or erythrocytes (Ho and Springer, 1983). Although the Mac-3 mAb also may label dendritic and endothelial cells, strict gating criteria were employed to exclude these other cell populations from analyses. Flow cytometric analysis of M is demonstrated in Fig. 3. The dot plot of FSC versus SSC initially was used to depict the whole PEC population (Fig. 3A). Region R2 was drawn to enclose the larger-sized (based on FSC) cells of interest. Similar regions designated R3, R4, and R7 (Fig. 3, B, C, and D, respectively) were drawn to enclose the cells of interest using dot plots of cellular size (FSC; Fig. 3B) or granularity (SSC; Fig. 3C) versus fluorescein fluorescence (FL1), as well as fluorescein versus PE fluorescence (Fig. 3D). Mutually exclusive addition of the cellular events located within regions R2, R3, R4, and R7 then was performed using CELLQuest software. The resultant group of cellular events located within region R4 was designated area G6 (Fig. 3E). The cells within G6 preferentially labeled for the Mac-3-FITC mAb, further confirming their identity as M. Once G6 was defined using Mac-3-FITC labeling of M, all subsequent samples were analyzed using the same regions.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Flow cytometric analysis of PEC cells demonstrating selective M gating. Dot plots of FSC versus SSC (A), FSC versus log-FITC fluorescence (B), SSC versus log-FITC fluorescence (C), and log-FITC versus log-PE fluorescence for PEC (D) are shown. Each dot represents a single cell; 25,000 cells are represented in each plot. The lower-end cutoff on the FSC plots indicates the threshold cutoff, which was set to eliminate lysed red blood cells and cellular debris from analysis. Mutually exclusive addition of the cellular events (E) located within regions R2, R3, R4, and R7 indicated in A-D above using CELLQuest software results in the cellular events viewed within R4 (E). Compare with R4 in C. This new region, G6 = R2+R3+R4+R7, was used to identify the M populations for analysis of labeling, because cells within this region also labeled preferentially with the Mac-3 mAb.

View this table: [in this window] [in a new window]   TABLE 3 Mouse peritoneal M labeled for the -opioid receptor Resident and TG-elicited peritoneal M, gated based on their distinctive FSC versus SSC cellular characteristics, as well as on their labeling with the anti-Mac-3 mAb, were analyzed for -opioid receptor expression as described in Materials and Methods and as reported previously (Ignatowski and Bidlack, 1998). Data are the mean ± S.E. of four or six separate experiments, each performed in triplicate.

Magnitude of -Opioid Receptor Expression on Immune Cells. -Opioid receptor labeling of resident, peritoneal M next was compared with that of the primary lymphocytes, as well as with that of the R1EGO thymoma cell line. It was found that the magnitude of labeling was much greater on M than on lymphocytes (Table 4). Specific labeling was determined by subtracting nonspecific from total labeling. The subsequent differences in labeling are evident by comparing the median PE fluorescence intensity values for specific labeling among the immune cell populations of interest (Table 4). The greatest magnitude of labeling is demonstrated by the R1EGO positive control cell line, which is rich in -opioid receptors, followed by that demonstrated by resident M. Compared with lymphocytes, M demonstrate approximately 25-fold greater labeling for the -opioid receptor. Although PE arbitrary fluorescence intensity units cannot be used to specifically state the number of receptors per cell, using this amplification-labeling procedure, the difference in magnitude of labeling suggests that resident, peritoneal M express more -opioid receptors per cell than either splenocytes or thymocytes.

View this table: [in this window] [in a new window]   TABLE 4 Magnitude of -opioid receptor labeling on various immune cell populations -opioid receptor labeling was performed as described in Materials and Methods.

In Vivo LPS Activation of Resident, Peritoneal M. Based on the findings that -opioid receptor expression was different on TG-elicited M compared with resident M and that in vitro LPS stimulation did not alter the lack of labeling on the TG-elicited M, we next decided to investigate the effect of LPS activation on nonelicited, resident M -opioid receptor expression. In vivo M stimulation was chosen as an alternative to in vitro stimulation because of the large quantity of mice necessary to undertake the in vitro experiments. For these studies, the concentration of LPS (10 µg) chosen for i.p. injection into mice was based on reports of similar, nonlethal concentrations for in vivo M activation experiments, where cytokine production, a hallmark activation-inflammatory response of M, or thrombin receptor binding was monitored (Remick et al., 1989; Fisker et al., 1992; Wollenberg et al., 1993). Proper injection of LPS was determined based on the physiological observations of diarrhea and lethargy in LPS-injected mice compared with vehicle-injected mice as well as with noninjected mice. Determination of M -opioid receptor expression was undertaken 1 h (to assess immediate activation effects) and 24 h (to allow for protein synthesis or turnover) after LPS or saline control injections, as well as on resident, peritoneal M (noninjected). Comparison of -opioid receptor labeling on M from the three groups revealed no apparent difference in the expression of this particular opioid receptor at 1 h (specific labeling: control, 64 ± 3%; saline, 59 ± 5%; LPS, 52 ± 9%; and the percentage of M labeled for the -opioid receptor: control, 69 ± 6%; saline, 60 ± 6%; LPS, 55 ± 5%; n = 4) or at 24 h (data similar to that reported for 1-h post-LPS stimulation).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present report, flow cytometry combined with indirect immunofluorescence was used successfully to identify the -opioid receptor on subpopulations of lymphocytes and M. These findings substantiate further: 1) that -opioid receptors are not restricted to the central nervous system, 2) that this receptor type may be widely distributed on various immune cell populations, and 3) a role for -opioids in immunomodulation, as suggested by numerous functional studies (Foster and Moore, 1987; Taub et al., 1991; Ichinose et al., 1995; Radulovic et al., 1995).

Previous investigations in our laboratory have demonstrated specific -opioid receptor labeling of immature thymocytes, as demonstrated by the lack of inhibition for FITC-AA/PE labeling by both µ- and -selective opioids (Ignatowski and Bidlack, 1998). This specific labeling, which was inhibited by the -selective antagonist nor-BNI, also has been evidenced on the R1EGO thymoma cell line (Lawrence et al., 1995a; Ignatowski and Bidlack, 1998), as well as on human microglia (Chao et al., 1996). The present findings of -opioid receptor expression on mature, splenic lymphocytes and on resident, peritoneal M substantiate further this sensitive methodology for the detection of cellular receptors, which may be expressed at low levels and are not detectable by other techniques, such as radioligand binding.

The relative number of mature, splenic T-helper, T-cytotoxic, and B lymphocytes expressing the -opioid receptor was minor (23, 17, and 16%, respectively) when compared with the number of immature thymocytes (60%), demonstrating labeling for this receptor (Table 2; Ignatowski and Bidlack, 1998). These findings substantiate our previous reports of a concomitant decrease in -opioid receptor expression on cellular and phenotypic maturation of immature thymocytes to those cells that have up-regulated the expression of the CD3 antigen (Ignatowski and Bidlack, 1998). Studies in progress are designed to assess the effect of various T cell stimulatory agents on -opioid receptor expression.

Resident, peritoneal M, another immune effector cell population, similarly demonstrated specific -opioid receptor labeling (Table 3). Because the number of resident M obtained from the mouse peritoneum was very small (i.e., approximately 1 × 10⁶ cells/mouse) and the number of M necessary for -opioid receptor labeling was large (i.e., approximately 1.5 × 10⁷ cells), an i.p. injection of 3% sterile, aged thioglycollate broth was used to induce an acute inflammatory response, thereby increasing the number of M harvested from the mouse peritoneal cavity. However, TG-elicited M differed vastly from resident, peritoneal M. Mac-3 mAb labeling was more pronounced, both in the number of M expressing the antigen and in the density of antigen present per cell, on TG-elicited M as opposed to resident M. This finding is similar to that demonstrated by other preferential M mAb markers, such as the Mac-2 mAb. Although these mAbs do not label all resident M, they label elicited M to a greater extent, because these cells have up-regulated antigen expression (Ho and Springer, 1983; Melnicoff et al., 1989). Likewise, the two M groups differed in their expression of the -opioid receptor. Resident, nonelicited M demonstrated specific labeling for this receptor, similar to that demonstrated by lymphocytes (Table 4), whereas TG-elicited M lacked detectable -opioid receptor labeling (Table 3). As an attempt to induce -opioid receptor expression, TG-elicited M were stimulated in vitro with LPS at a concentration determined previously to activate M (Ignatowski and Spengler, 1995). However, in vitro LPS stimulation was unable to induce receptor expression in TG-elicited M after either 1, 14, or 24 h of incubation. Therefore, these findings suggest that: 1) the TG injection may have caused a down-regulation of the -opioid receptor on M, possibly because of a component of the TG, such as agar, which has been observed to be taken up by M harvested after TG injection (Eichner and Smeaton, 1983), or 2) the injection of TG elicited a population of M lacking in -opioid receptor expression to the peritoneal cavity. These elicited M may represent an immature M population recruited from monocyte influx or from another possible peritoneal M precursor population, such as omental M (Melnicoff et al., 1989; Wijffels et al., 1992). The recruited M have been shown in labeling and tracking studies to migrate to the site of acute, sterile-induced peritonitis on the departure of resident, peritoneal M (Melnicoff et al., 1989). Caution, therefore, must be used when disclosing and comparing results from studies employing TG as an eliciting agent for M enrichment, because elicited M differ from resident M in many ways, including metabolic (Morahan et al., 1982; Eichner and Smeaton, 1983) and bactericidal activity (Baker and Campbell, 1980).

In vivo administration of LPS was chosen to compare the effects of M elicitation (TG injection) with M activation (LPS stimulation) for -opioid receptor expression. Because TG is not considered a classic activator of M, but only an elicitor of M, a partial activator of M, or a priming agent for subsequent M activation (Karnovsky and Lazdins, 1978; North, 1978), it was of interest to determine whether LPS, a M-activating agent (Doe and Henson, 1978; Doe et al., 1978; Karnovsky and Lazdins, 1978), administered in vivo would affect resident M -opioid receptor expression in a manner similar to TG administration. Resident M stimulated in vivo with LPS for either 1 or 24 h did not appreciably alter their expression of the -opioid receptor. The complexity of interactions occurring during the in vivo response to an inflammatory stimulus, such as LPS, may account for this seemingly lack of response. It has been established that M and lymphocytes, in an area of localized inflammation, release a multitude of mediators including cytokines and endogenous opioids such as -endorphin, enkephalin, and dynorphin (Schafer et al., 1994). These opioids then may interact with multiple opioid receptors to induce analgesia at nerve terminals and to either enhance or suppress various immune responses. The lack in change of -opioid receptor expression on M after low-dose LPS stimulation in vivo suggests that the potential interplay among the various mediators (Schafer et al., 1994; Alicea et al., 1996) allows for the continual expression of this receptor throughout the 24-h time period assessed. Studies have demonstrated the beneficial, immunoenhancing regulatory effects of -opioid agonists on M functions, such as phagocytosis (Ichinose et al., 1995), as well as suppressive regulatory effects, such as inhibition of cytokine production by LPS-stimulated M (Alicea et al., 1996). Because cytokines have been shown to induce the release of endogenous opioids from M (Schafer et al., 1994), the latter findings support a possible feedback regulatory loop allowing for the maintenance of -opioid receptor expression during an acute inflammatory response.

Finally, as depicted in Table 4, the magnitude of specific -opioid receptor labeling on resident M was much greater than that determined for lymphocytes. This finding suggests that per cell, M express more -opioid receptors than lymphocytes. The endogenous -opioid peptide dynorphin suppresses certain T cell activities (Prete et al., 1986) while enhancing various M functions (Foster and Moore, 1987; Ichinose et al., 1995). These findings illustrate the preferential modulatory effect of this endogenous opioid on lymphocytes and M. This disparity in -opioid-mediated regulation of immune cells may relate directly to the differential expression of this receptor population on these cells.

In conclusion, we demonstrated the differential expression of the -opioid receptor on various immune cell populations. Interestingly, the population of primary immune cells that demonstrated the greatest magnitude of labeling for the -opioid receptor was the resident M. Numerous studies have demonstrated -opioid-induced regulation of cytokine release (Alicea et al., 1996), phagocytosis (Ichinose et al., 1995), and tumoricidal activity (Foster and Moore, 1987) by M, corroborating a functional role for this opioid receptor in mediating immunocompetence. Further understanding of the immune cells expressing the various opioid receptors will help to improve our understanding of the immune and inflammatory responses regulated, both directly and indirectly, by opiates.