We all are very familiar with the effects of cannabinoid receptor stimulation on the body. Relaxation, pain relief, and increased appetite probably come first to mind. These psychoactive effects result from activation of the CB1 receptor found on cells in the brain by tetrahydrocannabinol (THC).
But there is another receptor, called CB2, that can bind THC and other natural ligands for the cannabinoid receptor. The CB2 receptor is found on cells comprising the immune system and have a multitude of anti-inflammatory and immunosuppressive effects upon activation. In most cases, immunosuppressive effects are undesirable, but sometimes that can be beneficial. Today’s article is an example.
New research from the lab of Dr. Guy Cabral at Virginia Commonwealth University shows that stimulation of the CB2 receptor on macrophages inhibits migration of healthy immune cells towards the HIV Tat protein. Tat is an essential viral regulatory protein used by HIV to stimulate inflammatory responses and wreak havoc in the body. Tat protein looks suspiciously like some of our own chemokine proteins and can bind to a variety of receptors on immune cells causing activation of cascades that lead to migration of uninfected macrophages towards the HIV infected cells.
The paper by Erinn S. Raborn and Guy A. Cabral, published in the January 2010 issue of the Journal of Pharmacology and Experimental Therapeutics is titled: Cannabinoid Inhibition of Macrophage Migration to the Tat Protein of HIV-1 is Linked to the CB2 Cannabinoid Receptor. The authors used a macrophage cell line in a migration model system to demonstrate very specifically that when the CB2 receptor is stimulated, macrophages no longer respond to the Tat protein. The chemoattractant effects are abolished.
Here is an summary of their work.
Macrophages are the primary target for HIV infection and once infected, cells begin producing viral Tat (trans activating factor) protein and GP120 protein in addition to stimulating the production of cellular cytokines and chemokines that induce changes in the immunoregulation of the host. The HIV Tat protein has an additional role of acting as a potent chemoattractant for monocytes, thus contributing to the spread of infected cells.
Most drugs of abuse (opiates, cocaine, amphetamines and cannabinoids) have an adverse effect on immunity, increasing susceptibility to infection. The cannabinoids in particular have been shown to have anti-inflammatory properties, downregulating some of the chemokines and cytokines involved in stimulating macrophage to migrate to infections and inhibiting macrophage function. Cannabinoids have also been shown to down-regulate the expression of chemokine receptors, notably CCR5, one of the co-receptors used for HIV entry into cells. Thus, a link between the potential anti-HIV effects of the CB2 cannabinoid receptor has been established.
The purpose of this study was to determine whether cannabinoids exert any effect on the chemoattractant properties of Tat in macrophages. In the presence of cannabinoid agonists delta-9- tetrahydrocannabinol and CP55940, human macrophage-like cells (U937 cells) were inhibited from migrating towards Tat protein and this effect was due specifically to CB2. The results show a clear link between CB2 and the ability of macrophages to respond to the HIV protein in a cell culture system. This work provides the basis for a novel therapeutic target for preventing or reducing HIV associated immunopathology and dissemination in vivo.
Materials and methods:
Cells: The human leukemic monocyte cell line U-937 was used.
Drugs: CB1 and CB2 receptor agonists used were: THC and CP55940. The CB2 specific agonist was O-2137-2 and the CB1 specific agonist was ACEA. The CB1 and CB2 receptor antagonists were SR141716 (SR1) and SR144528 (SR2), respectively. The full names of the drugs and the Ki information is described in the paper.
Tat: Recombinant human HIV Tat protein was obtained from Immunodiagnostics, Inc.
Cell Migration Assay: 35 mm tissue culture plates with upper and lower compartments separated by a polycarbonate 8 micron pore membrane were used. Drug treated or control treated U937 cells were incubated on the top chamber and Tat protein or serum-free media plus vehicle was in the bottom chamber. Migration of cells to the bottom chamber was visualized with an Olympus CK2 inverted microscope connected to a digital video camera. The number of cells were manually enumerated. A greater than 2-fold increase in the number of cells in the presence of chemoattractant compared to no Tat was a positive response. The EC 50 or inhibitory concentration was the concentration of cannabinoid that results in a 50% reduction in macrophage migration.
Knockdown of CB2 expression using siRNA and RT-qPCR: siRNA for the CB2 receptor was used for transient transfection of cells and knock-down shown using Western blot analysis. SYBR Green was used for qPCR analysis after reverse transcription of RNA, qPCR was performed using the SmartCycler. Full details of the experimental design for qPCR and transfection are provided in the paper.
To begin, the authors first confirmed the expression of the CB2 receptor in U937 cells, both on the RNA and protein level. The CB1 receptor, typically expressed in cells of the brain, was not found in U937 cells using RT-qPCR. Their second set of confirmatory experiments proved that the U937 cells would migrate in response to Tat protein as has been previously described for human monocytes in blood. The migratory response was maximum at 50 nM Tat and so the authors used this concentration for their studies.
The next experiments looked at the migration of macrophages in the presence of the different drugs. All results were compared to vehicle controls (ethanol was used to dilute the drugs). Using vehicle alone with Tat protein in the bottom compartment, migration was the same as with no vehicle. When cells were treated with THC, however, migration of macrophage was inhibited by 50%. And using the agonist CP55940, migration was inhibited by 58%. Using the CB2 receptor specific agonist, the same effect of >50% inhibition was observed, however, but not suprisingly, using a CB1 receptor agonist, ACEA, had no effect on migration.
These results clearly indicate that the CB2 receptor on the human macrophage-like U937 cells plays a role in migration in the presence of the HIV Tat protein and this effect can be blocked when the receptor is activated.
To confirm the effect of migration was due to the CB2 receptor, the receptor antagonists were used to demonstrate that when signalling through the receptor is blocked, the effect is reversed. The antagonist will bind the receptor but not activate the signalling cascade in the cell, thus blocking it from being activated by the ligand CP55940. Using the CB2 specific antagonist SR2 alone, migration in the presence of Tat is the same as controls- it is not inhibited. When CP55940 is combined with the SR2 compound, inhibition of migration is reversed. SR2 prevents the protective anti-migration effect of CP55940.
To further prove the role of CB2 in this effect, siRNA mediated CB2 knockout cells were employed in the cell migration assay. The authors confirmed that neither the transfection reagent nor the siRNA itself had any effect on cell migration. Using the CB2 knockout cells in the presence of THC, migration was observed similarly to untreated cells. This is consistent with the results of blocking the CB2 receptor with antagonist. The CB2 receptor was not available for activation by the THC and thus no protective anti-migration effect was observed.
Chemokines are cytokines that function by directing the flow of inflammatory cells to sites of injury or infection in the body. HIV uses our immune system cascade meant to protect us for its own benefit, by producing the viral Tat protein to bind the receptors meant for chemokines and stimulate the migration of more healthy macrophages to the HIV infected cells, increasing their opportunity to spread. The stimulation of chemokines also increases the number of receptors on the surface, making it easier for HIV to infect new cells.
The ability to slow down the migration of healthy cells towards HIV infected cells and to down regulate the expression of chemokine receptors would slow the progression of HIV associated disease. In this paper, the authors demonstrate a connection between the cannabinoid receptor on immune cells, CB2, and the migration of macrophages towards Hiv Tat protein. When the CB2 receptor is stimulated with an agonist, migration is inhibited. Whether this is due to down regulation of the chemokine receptors used for binding or due to down regulation of chemokines from CB2 activated cells has yet to be determined.
But the fact remains that the cannabinoid receptor CB2 may be a therapeutic target for preventing hyperactivation of the immune system by HIV and potentially, in the future, to help stop widespread infection.
I found this paper to be an exciting advance in the fields of HIV and drugs of abuse. A therapy involving cannabinoids would be far less toxic than current drugs used to stop the progression of HIV. With all the ways HIV has found to use our own immune system against us to survive, an approach that counteracts HIV by taking back control of the immune system could be far more effective than developing toxic drugs that target HIV directly or using cytokines that further activate a tired immune system.