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. Author manuscript; available in PMC: 2013 Feb 6.
Published in final edited form as: J Neuroimmune Pharmacol. 2010 Dec;5(4):546–552. doi: 10.1007/s11481-010-9209-8

In vivo morphine treatment synergistically increases LPS-induced caspase activity in immune organs

Michael Olin 1, Brian Lee 2, Sabita Roy 3, Thomas Molitor 4,*
PMCID: PMC3565582  NIHMSID: NIHMS436290  PMID: 20390371

Abstract

Apoptosis is an important mechanism for the elimination of infected cells, which would normally serve as hosts for further pathogen replication. Apoptosis is initiated through complex pathways involving a family of cysteine proteases known as caspases. The detection of apoptosis is essential for understanding the long-term health effects inflicted by the therapeutic use of opiate drugs such as morphine for pain treatment following major trauma or disease and abusive use of such drugs in addiction. Common practices of apoptosis detection involve the removal of tissues, which subsequently induce spontaneous apoptosis unrelated to the actual effects of the opioid drug exposure. The objective of this study was to develop an in vivo detection method for assessing morphine’s ability to directly induce apoptosis, and in the combination of morphine following an inflammatory response induced by lipopolysaccharide (LPS). Mice were administered saline, morphine, LPS, or a combination of morphine and LPS. Prior to sacrifice, mice were injected with a poly-caspase-specific apoptosis detection probe, to detect internal caspase activity in vivo. Administration of morphine alone did not directly induce apoptosis. However, morphine significantly enhanced the LPS induced apoptosis in splenocyte and bone marrow cells as well as in spleen, liver, and thymus tissues. The use of a poly-caspase detection probe methodology, to label apoptotic cells in vivo, provides a powerful quantitative tool for the in vivo analysis of caspase activity.

Introduction

Morphine treatment is one of the most widely utilized strategies for the control of chronic pain world-wide (Portenoy and Lesage, 1999). The cytotoxic effects of opiates, however, remain a major concern. In vitro studies have indicated that morphine induces apoptosis in a variety of human and rodent peripheral immune system-related cells (Singhal et al, 1998; Yin et al, 1999), as well as embryonic neuronal and glial cultured cells (Hu et al, 2002). Morphine induces oxidative stress in macrophages, T lymphocytes, and hepatocytes inducing apoptosis via a Fas receptor promoting the analgesic effects of opiates and manifesting secondary cytotolytic effects involved in pain treatment regimens (payabvash 2006, Wang 2001).

The potential neurotoxic effects of chronic opioid administration have been studied in multiple in vitro and in vivo models. In selected studies morphine’s induction of apoptosis was associated with an up-regulation of the pro-apoptotic caspase-3 and Bax proteins and down-regulation of the anti-apoptotic Bcl-2 protein (Mao et al, 2002). However, others reported that morphine mediated apoptosis via the Fas /FasL extrinsic pathway signal transduction as the principle mechanism that initiates apoptosis following morphine administration (Yin et al, 1999; Boronat et al, 2001, Emeterio et al, 2006).

The detection of apoptotic events require sex vivo sample harvesting techniques such as DNA fragmentation assays utilizing the TUNEL assay (Duan et al, 2003), or extracellular staining by Annexin (Liu et al, 2001, Stahelin et al, 1998). Subsequent in vitro apoptosis methods were developed employing fluorogenic/chromogenic caspase probes (Lee et al, 2003, Mizukami et al, 1999), detecting active caspase enzymes by immunocytochemistry or their specific cleavage products (Li et al, 2000) and mitochondrial potentiometric dye based assays (Pootet al, 1996, Salvioli et al, 1997). More recently, fluorescence labeled inhibitors of caspases (FLICA) (Smolewski et al, 2001)probes were developed and employed for the detection of apoptosis in vitro. FLICA reagents employ a cell permeable probe that covalently binds to active caspases. In vitro apoptotic detection by FLICA reagent demonstrated a strong correlation with TUNEL (r =0.95) (Bedneret et al, 2000).

FLICA probes provide a powerful advantage over other apoptosis detection systems. Conventionally apoptosis detection requires removing tissues from their natural environment thereby depleting the individual cells of required oxygen and nutrients and inducing apoptosis (Malhotra et al, 2001). In addition, mechanical manipulation of tissue physically damages cell membranes leading to increased levels of cellular death through apoptosis as well as necrosis. The increased levels of apoptosis would not be distinguishable from apoptosis induction resulting from the experimental agent resulting in the generation of false positives. The study presented here was designed to eliminate the need for ex vivo staining through the use of FLICA probes modified for in vivo caspase detection. In this study morphine significantly enhanced LPS induced apoptosis in various tissues. By employing the cell permeant probe for in vivo caspase labeling, we were able to circumvent the false positive results due to tissue manipulation.

Materials and methods

Mice

C57BL/129 wildtype and opioid receptor μ-knockout mice were housed under specific pathogen free conditions in accordance with the University of Minnesota Institutional Animal Care and Committee.

Morphine and LPS injection of mice

Mice were placed into placebo, morphine, LPS, or morphine +LPS treatment group, n=8/group. Morphine was administered via intraperitoneal (IP) injection at a daily dosage of 25 mg/kg 24 h and 48 h prior to sacrifice. Twenty-five (25) μg (1 mg/kg) of LPS purified from E.coli(strain ???) was administered intraperitoneal 24 h prior to euthanasia. Saline was used as a placebo control

In vivo Apoptosis detection

Thirty min prior to sacrifice, 10 μg of multi-caspase detection probe (FLIVO) (Immunochemistry Technologies, LLC. Bloomington, MN) was administered via tail vein injection at a dosage of 400 μg/ kg. FLIVO injection solution was prepared by dissolving the contents of one vial of the carboxyfluorescein – valylalanyl aspartic acid-fluoromethyl ketone (FAM-VAD-FMK) caspase detection reagent in 50 μL of tissue culture grade DMSO. The FLIVO reagent was further diluted 1:10 in sterile isotonic injection buffer to yield the 1X FLIVO reagent solution. A 100 μL volume of FLIVO caspase probe was injected via tail vein.

Splenocytes and Bone Marrow preparation

Mice were CO2 anesthetized followed by cervical dislocation. Spleens were removed and washed three times in 1X PBS buffer solution. Splenocytes were isolated from the spleens as previously described (Lin et al, 2007). Spleens were homogenized and strained through a 100 μm pore size cell straining filter to remove connective tissue. Splenocytes were centrifuged at 650 x g for 5 min, and resuspended in 1 mL deionized H2O to lyse remaining red blood cells (RBC’s), washed and analyzed by flow cytometry. Bone marrow isolation was accomplished by removing skin and muscle from hind legs using aseptic technique. Femurs were severed between joints. Bone marrow was flushed by inserting a 25-Ga needle into the cavity and injecting sterile cold phosphate-buffered saline solution containing 2% fetal bovine serum into the cavity to remove the cells (Aldegueret al, 2002 ). Bone marrow wash medium was collected into sterile 50 ml tubes and kept on ice. Residual RBC’s were water lysed, and the bone marrow contents were immediately analyzed by flow cytometry.

Tissue isolation

Spleen, liver, thymus, and central nervous system tissues were snap frozen in liquid nitrogen, and placed in plastic trays, immersed in OCT, and stored frozen at −80°C. Frozen tissues were subsequently sectioned in low light conditions and mounted on a glass slides for fluorescent microscopy.

Immunohistochemistry

Tissue sections were mounted on glass slides and analyzed by fluorescent microscopy using a FITC filter. Dapi staining was used to determine cell associated apoptotic events. Sections were photographed and analyzed by MetaMorph analysis (Molecular Devices Corporation, Sunnyvale, CA.) program for apoptosis positive cell counts. Ten sections were analyzed at a magnification of 60X.

Data analysis and Statistical analysis

Splenocyte and bone marrow apoptosis were analyzed at final concentration of 1 x 10 6 cells/mL using a FACS caliber, results were analyzed by CellquestPro (Becton Dickinson Immunocytometry System, San Jose, CA). Data was analyzed using a dot plot separated into four quadrants. Percentages of cells killed by cytotoxic activity were calculated by dividing the events in the (upper right quadrant) / (upper and lower right quadrants) X 100 and then subtracting the percentage of spontaneous cell death from target-cells-only control tube. Statistically significant differences were assessed using unpaired Student’s t-test for analysis between two groups. Differences between data were considered statistically significant at p<0.05.

Results

Morphine and LPS synergy in spleenocyte apoptosis induction

There are many commercially available assays for the detection of apoptosis, each having particular strengths and weaknesses. The weakness of current apoptosis detection methodologies is the requirement for ex vivo manipulation of tissues. Recently, a cell-permeant fluorescently labeled probe was developed to detect and quantitate apoptosis via caspase activity (Lee et al, 2003). The present study is the first to use a cell permeant probe to label, in vivo, cells undergoing apoptosis in mice. Thirty min post inoculation, mice were euthanized; splenocytes were immediately purified and analyzed for apoptosis using flow cytometry. The probe labeled the apoptotic cells in vivo not via ex vivo manipulation. Using CellquestPro, a histogram of the various treatment groups were derived and overlaid for analysis (Fig 1a). The percentages of apoptotic cells were imported into a dot plot (Fig 1b). LPS administration into mice significantly induced apoptosis compared to placebo treated mice (p=0.03). Co-administration of morphine + LPS significantly increased the percentage of cells undergoing apoptosis (p=0.01). Morphine alone did not significantly induce apoptosis.

Figure 1.

Figure 1

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Administration of morphine significantly increased LPS apoptosis in cells of the immune system. Morphine was administered prior to LPS apoptosis induction. Caspase specific probes were injected into mice 30 min prior to sacrifice. Splenocytes from placebo, LPS, and morphine + LPS treated mice were purified and analyzed for apoptotic events. (a) Splenocytes from placebo, LPS, and LPS + morphine were purified and analyzed by flow cytometry. Using CellquestPro, a histogram of the various groups was overlaid to analyzed morphine effect on LPS induced apoptosis. Plots are representative of group n=8. (b) Percentages derived from CellquestPro were plotted in a bar graph. A statistically significant change determined by p<0.05

Synergistic effects of Morphine and LPS on the induction of apoptosis in bone marrow

The bone marrow is essential for the generation of progenitor cells, maintaining a constant percentage of competent immune components. To determine if acute morphine exposure enhances the apoptotic effect of LPS in progenitor cells, bone marrow was isolated from the various treatment groups and immediately analyzed by flow cytometry (Fig. 2). Percentages of apoptotic cells were imported into a dot plot (Fig. 2b). LPS exposed mice significantly induced an apoptotic response in bone marrow compared to placebo mice (p=0.02). Although morphine administration enhanced percentages of apoptotic cells in bone marrow, the increase of apoptosis observed was not statistically significant.

Figure 2.

Figure 2

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FLICA probe was administered into various mice groups 30 min prior to bone marrow collection. (a) Bone marrow from placebo, LPS, and morphine + LPS were purified and analyzed by flow cytometry. Using CellquestPro, a histogram of the various groups was overlaid to analyzed morphine effect on LPS induced apoptosis. Plots are representative of group n=6. (b) Percentages derived from CellquestPro were plotted in a bar graph. A statistically significant change determined by p<0.05

Synergistic effects of Morphine and LPS on apoptosis in tissues

A critical question that we set out to answer was, does an immune activating signal such as LPS act in concert with morphine in promoting apoptosis at the tissue level? To answer this question, spleen, thymus, CNS, and liver tissues were immediately removed from the different mice treatment groups following in vivo exposure to the FLIVO apoptosis probe, and snap frozen in liquid N2. These frozen tissues were sectioned and analyzed by fluorescence microscopy. In the spleen, thymus, liver, acute LPS exposure was found to induce apoptosis as detected by fluorescence microscopy( Fig 3). The co-administration of morphine and LPS significantly increased percentages of apoptotic cells in spleen (p =0.01), thymus (p = 0.01), and liver tissue cells (p=0.02) when compared to LPS-only treated mice. There was no significant increase of apoptosis in the CNS (Table 1). Morphine exposure alone did not significantly induce apoptosis compared to placebo (Table 1).

Figure 3.

Figure 3

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Number of apoptotic cells in the thymus is increased in tissues following morphine and LPS administration. Mice were administered placebo (a), LPS (b), or morphine + LPS (c), 30 min prior to sacrifice; FLICA apoptosis probe was administered via tail vain. Tissues were frozen, dapi stained, and analyzed by fluorescence microscopy.

Table 1.

Morphine’s effect on apoptosis in tissues.

Tissues Treatments % Positive Animals Counts
Spleen Placebo 25 26
Morphine 50 39 ± 12
LPS 75 48 ± 6
Morphine + LPS 100 117 ± 23*
Liver Placebo 25 11
Morphine 75 12 ± 3
LPS 75 17 ± 5
Morphine + LPS 100 82 ± 16*
Thymus Placebo 0 0
Morphine 50 12 ± 6
LPS 50 21 ± 7
Morphine + LPS 100 103 ± 13*
CNS Placebo 0 0
Morphine 0 0
LPS 25 6.5
Morphine + LPS 50 22.5 ± 28
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*

Denotes a statistically significant difference between LPS and morphine + LPS treatment groups

Synergistic effects of Morphine is MOR mediated

To address the question of the specificity of morphine’s in vivo effect on apoptosis, splenocytes from μ-opioid receptor knockout (MORKO) mice were compared to wildtype mice for apoptosis. Splenocytes from the LPS + morphine or saline-negative control mice treatment groups were harvested and analyzed by flow cytometry (Fig. 4a). Percentages of apoptotic cells were imported into a dot plot (Fig. 4b). Morphine significantly enhanced LPS induced apoptosis in wildtype mice. In comparison, apoptosis was significantly reduced in MORKO mice treated with morphine and LPS (p=0.01). Morphine alone did not significantly induce apoptosis in either wildtype or MORKO mice.

Figure 4. Increase in morphine apoptosis is mu-receptor mediated.

Figure 4

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To determine if the increase in apoptosis is mu-receptor mediated, mu-knockout and wildtype mice were compared for the induction of apoptosis following LPS administration. Mu-knockout and wildtype mice were administered morphine + LPS. Placebo was administered in mu-knockout for a negative control. (a) Using CellquestPro, a histogram of the various groups was overlaid to determine if increased apoptosis is morphine mediated. Plots are representative of group n=8. (b) Percentages derived from CellquestPro were plotted in a bar graph. A statistically significant change determined by p<0.05

Discussion

Morphine is one of the mostly widely prescribed analgesic opioids used for the treatment of pain. Opioid drugs have consistently proven useful in the management of chronic cancer pain and have been classified by the World Health Organization as the second step in the analgesic ladder for pharmacotherapeutic treatment of long term pain (Portenoy and Lesage, 1999, Radbranch et al, 1996). Unfortunately, high dosage of morphine administration has been associated with varying levels of adverse cytolytic effects (Nelson et al, 2000).

The experimental design of this study was to pursue an acute, rather than chronic morphine exposure model. In contrast to the chronic morphine exposure model, which was well documented to cause a high incidence of apoptosis induction following a stimulis, the induction of apoptosis due to acute morphine exposure remains unknown. Oxidative stress has been associated with an induction of apoptosis, associated with acute and chronic morphine treatments in various cell types such as cells of the immune system (Di Francesco et al, 1998), kidneys (Patel et al, 2003a; Patel et al, 2003b), epithelial cells Macchia et al, 1999), CNS(Goudas et al, 1999; Hauser et al, 1998) and liver (Payabvash et al, 2006). To determine “in vivo” morphine’s effect on caspase induction, mouse splenocytes, bone marrow, thymus, CNS, spleen, and liver tissues were analyzed using the in vivo caspase detection format. In the present study, morphine alone did not significantly induce apoptosis in any of the tissues examined. However, acute morphine administration in combination with LPS administration induced a highly significant increase in apoptosis in multiple tissues.

The immune activating signal LPS has been well documented to induce apoptosis. Using cell membrane associated signal transduction mechanisms such as TLR-4 and TNFα, the pro-inflammatory mediator, LPS, can activate extrinsic (caspase 8) apoptotic pathways. Intraperitoneal LPS injection is often used as a model system for simulating the gram negative, endotoxigenic, and bacterial sepsis state. Previous studies have concluded that morphine augments the effects of LPS in mouse thymus and macrophage cells (Roy et al. 1999), mouse leukocytes (Ocasio et al, 2004), and human and rat vascular endothelial cells (Liu et al, 2004). Prior chronic morphine treatment leads to the enhancement of LPS induced septic shock associated with an elevation in the inflammatory cytokines (TNFα, IL-1β, and IL-6) following exposure to an LPS challenge (Ocasio et al, 2004). Based upon this previously documented synergism, it was hypothesized that morphine would significantly increase LPS induced apoptosis within a mouse acute morphine treatment model system, providing a clinically significant model system for demonstrating the utility and assay sensitivity of an in vivo apoptosis detection assay format. The results of this study coincide with the hypothesis that acute morphine administration increases LPS induced apoptosis. In mice treated with morphine and LPS, there was a markedly increased amount of apoptosis detected in the spleen, liver, and thymus.

Although there has been an abundance of studies reporting that the induction of the apoptosis cascade is via morphine’s interaction with morphine receptors (MOR); the mechanisms involving the induction of apoptosis remain controversial. MOR is located on a number of different cell types including neurons and microglia (Hu et al, 2002), lymphocytes (Wang et al, 2001), endothelial cells (Liu et al, 2004), spleen, heart, and lung tissue (Yin et al, 1999). To address the question whether morphine induced apoptosis was opioid receptor mediated; mu-knockout mice were analyzed for apoptosis induction following acute morphine exposure. The study presented reported a clear decrease in the number of cells undergoing apoptosis in mu-knockout mice compared to wild type animals suggesting morphine modulates apoptosis vis the mu-receptor.

Studies have eluded that morphine administration induces apoptosis, via either Fas receptor or mu-receptors. However, other studies failed to see a significant increase in apoptosis due to morphine alone. Apoptosis is an important mechanism involved in the removal of infected or dying cells. However, the induction of apoptosis involves removal of tissues capable of inducing apoptosis leading to false positive results. The study presented is the first to use in vivo caspase detection assay to study the effects morphine has on the induction of apoptosis. By detecting the in vivo caspase activity, the study presented concludes that acute morphine treatment alone has minimal effect on the induction of apoptosis. However, co-administration of both LPS and morphine significantly enhanced apoptosis in all cell and tissue types with the exception of extracted bone marrow. By injecting the caspase probe directly into the animals, unwanted generation of sample manipulation associated apoptosis was avoided. Reduction of this background apoptosis detection level would be expected to provide a more accurate measurement of the true morphine and morphine + LPS associated apoptosis generation

Morphine induced apoptosis has remained controversial for many years. Studies have eluded that morphine administration induces apoptosis, however, other studies failed to detect a significant increase in apoptosis following morphine administration. Apoptosis is an important mechanism involved in the removal of infected or dying cells, false positive results due to manipulation of tissues makes it difficult to accurately interpret experimental results. The study presented is the first to use in vivo caspase detection assay to study the effects morphine has on the induction of apoptosis. Using in vivo caspase activity, we found that acute morphine treatment alone has minimal effect on the induction of apoptosis. However, co-administration of both LPS and morphine significantly enhanced apoptosis in all cell and tissue types with the exception of extracted bone marrow. By injecting the caspase probe directly into animals, study complications due to unwanted apoptosis due to sample manipulation was avoided. In conclusion, the use of the novel in vivo caspase detection method allowed us to conclusively determine that although morphine alone failed to induce apoptosis, morphine significantly increased LPS induced apoptosis.

Acknowledgments

Special thanks to Rick Charboneau for his assistance in the laboratory. Michael Olin was supported by the National Institute of Health, National Research Service Award T32 DA07097 from the National Institute on Drug Abuse.

Contributor Information

Michael Olin, Email: olin0012@umn.edu, University of Minnesota. Center for Infectious Diseases and Translational Research Translational Research Facility, Minneapolis, MN 55455, 612-616-2246.

Brian Lee, Email: brian@immunochemistry.com, 952-888-8788. Immunochemistry Technologies, LLC, 9401 James Ave S. Bloomington MN, 55431.

Sabita Roy, University of Minnesota, department of Pharmacology, Minneapolis, MN 55417, 612-624-4615.

Thomas Molitor, University of Minnesota. Veterinary Population Medicine, 225 Veterinary Teaching Hospital, 1365 Gortner Ave., St. Paul, MN 55108.

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