 d posttreatment) of EAE. Non-EAE controls WT and Panx1 KO spinal cords displayed limited number of infiltrating CD4+ T lymphocytes. Error bars, SE. *p ≤ 0.01. N.S., no significance.
Discussion
We have previously shown that HIV binding to CD4 and CCR5/CXCR4 stimulated a biphasic (early, 5–60 min and late, 10–24 h) opening of Panx1 channels. However, the mechanism by which these channels are activated by HIV or other physiological stimulus is unknown. In this study, we show that physiological concentrations of chemokines, which bind CXCR4 and CCR5, induce a transient (5 min) activation of Panx1 channels on CD4+ T lymphocytes, which results in ATP release, FAK phosphorylation, cell polarization, and transmigration. We propose that blocking Panx1 channels could be a potential therapeutic target to reduce inflammation and transmigration of immune cells into compromised tissues.
Panx1 channels are usually closed to prevent unbalance of the electrochemical gradient across the plasma membrane (32). Currently, the mechanisms of Panx1 opening are poorly explored, but it has been suggested that one mechanism involves mechanical stimulation. An increase in fluid shear force such as the application of suction to a patch of membrane or physical stretching of the entire cell increases the permeability of the channel (8, 33–36). Panx1 channels have also been shown to open in response to increasing extracellular potassium concentration (18, 19, 37). Recent work has identified that proteolytic cleavage of Panx1 C-terminal tail by caspase-3 or caspase-7 results in ATP secretion, cellular recruitment, and inflammation in association with apoptosis (13, 38). As was previously described, opening of Panx1 channels results in changes in ionic gradients as well as the release of several factors, including ATP (38–42). ATP is released from intact apoptotic cells, serving as a danger signal that recruits phagocytic cells (13). In this study, we identified that SDF-1α/CXCL12 stimulation induces activation of Panx1 channels, which results in the release of ATP through the channel because blockade of Panx1 channels prevented ATP release.
Our data indicate that, in contrast to HIV infection, which results in a prolonged activation of this channel, chemokines that bind to CCR5 and CXCR4 induce a transient activation of Panx1 channels. In this regard, it has been shown that gp120 activates a different signaling pathway than the one induced by chemokines (43–50). Once HIV or specific chemokines bind to the chemokine receptor, activation of GPCR occurs, which results in the recruitment of the G protein complex inducing the exchange of GTP for GDP and causing the disassociation of the βγ dimer from the α unit, resulting in downstream signaling (51–53). In agreement, our data demonstrate that PT blocks the opening of Panx1 channels induced by SDF-1α/CXCL12 treatment. Evidence in the literature suggests that HIV-1 gp120 triggers Pyk2 phosphorylation in CD4+ lymphocytes. However, HIV-1 gp120 binding to CXCR4 fails to induce phosphorylation of ERK1/2 MAPK, even though SDF-1α/CXCL12 binding to CXCR4 clearly induces phosphorylation of ERK1/2 MAPK in CD4+ cells (47, 54). Chemokines are thought to bind to a pocket formed by the transmembrane helices found in G protein–coupled receptors, which allow a conformational change that promotes guanine nucleotide exchange in G proteins (55). Chemokines bind this pocket at their N termini; HIV envelope does not induce an activated conformation of CCR5 to enter and can bind away from the pocket. This suggests that some of the elements of the binding site are distinct between HIV and chemokines, which bind to the same receptors (45). Taking this evidence into consideration, the differential temporal activation of Panx1 channels seen in HIV infection and chemokine stimulation could be a result of differential downstream signaling cascade induced by different binding sites used by the virus and the chemokines.
In this study, we demonstrated that FAK is phosphorylated at Tyr576/577, Tyr925, and Tyr397 residues during SDF-1α/CXCL12 stimulation, and this phosphorylation is prevented when Panx1 channels are blocked as well as in CD4+ T lymphocytes from Panx1 KO mice. Phosphorylation and activation of FAK have been described to play an integral role in cytoskeletal rearrangement induced by G protein–coupled receptor stimuli such as CXCL12 (24, 25). On the basis of the present results and above considerations, it is proposed that Panx1 channels in concert with purinergic receptors induce the phosphorylation of FAK located at the leading edge of polarizing cell resulting in cell migration.
MS is an autoimmune inflammatory demyelinating disease characterized by destruction of myelin and migration of autoreactive T lymphocytes into the nervous system (56). In MS, SDF-1α/CXCL12 contributes to the transmigration of autoreactive T lymphocytes into the CNS and is elevated in inflamed endothelial cells, astrocytes, and cerebrospinal fluid of MS patients (29–31). The recent work of our collaborators has demonstrated that inhibition or deletion of Panx1 improved the neurologic and pathological symptoms of EAE (15). Our present work demonstrates that this delayed onset of clinical symptoms was due to a decreased migration of CD4+ T lymphocytes into the spinal cord. CD4+ Th1 and Th17 cells that migrate into the CNS induce EAE and CD4+ cells are significantly increased in lesions, serum, and cerebrospinal fluid of MS patients (57–59). Therefore, it is likely that clinical symptoms may be delayed by specifically targeting the T cell subset involved in the damage by controlling Panx1 channel activity to reduce transmigration of activated leukocytes into the CNS.
In conclusion, Panx1 channels are transiently opened in response to chemokine gradients, and an opening of this channel is required for the pathogenesis of several diseases such as HIV and MS. We propose that blocking these channels can be exploited as a therapeutic target to reduce the devastating consequences of several inflammatory diseases.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank the New Jersey/New York Blood Center.
Footnotes
This work was supported by National Institutes of Health-National Institute of Mental Health Grant MH096625, Public Health Research Institute funding (to E.A.E.), National Institute of Neurological Disorders and Stroke NS052245 (to E.S.) and T32 NS07439 (to S.E.L.), by National Multiple Sclerosis Society Fellowship FG2035A1 (to S.E.L.), an Alfred P. Sloan Foundation minority fellowship (to S.V.), and Mount Sinai NeuroAIDS Disparities Summer Institute Grant R25 MH080663 (to S.V.)
The online version of this article contains supplemental material.
Abbreviations used in this article:
- Apy
- apyrase
- EAE
- experimental autoimmune encephalitis
- Etd
- ethidium
- FAK
- focal adhesion kinase
- KO
- knockout
- mCD4
- mouse CD4
- MS
- multiple sclerosis
- oATP
- oxidized ATP
- Panx
- Pannexin
- Prob
- probenecid
- PT
- pertussis toxin
- SCR
- scrambled peptide
- siRNA
- small interfering RNA
- WT
- wild-type.
- Received November 18, 2015.
- Accepted March 9, 2016.
- Copyright © 2016 by The American Association of Immunologists, Inc.This is what I am tackling the western Massachusetts snow season with this winter. Wish me luck. The less snow the better.
Nice! That should be able to tackle most any snow storm with ease.
Nice set up....His and hers!
Well if you don't get enough snow over there, you can bring'em to South Eastern Mass and help me out! LOL..In this lecture, we are going to deploy the Configuration Manager client using the Client Push method.
Now, if you are working in a lab environment, make sure you have your Domain Controller, your SCCM, and your Workstation VMs all powered On before proceeding on to this lecture.
We are also going to be switching between the SCCM server and our Windows 10 VM quite frequently, so be prepared to do that.
Before we attempt a client push, we need to make sure that SCCM will use a Domain Administrator account to install the client. We can do this by navigating over to the Administration tab, and selecting Site configuration, Click on Sites, and right-click on our server, and Client Installation Settings, click on Client Push Installation.
It's going to open the Client Push Installation Properties.
We click on the Accounts tab and we can see that there’s no account listed here. So we are not going to be able to do any kind of Client Push because we are not specifying an account.
What we need to do is click this Star button up here and select New Account.
Even though we are not creating a new account, as far as the Configuration Manager is concerned, there is no account specified and we are going to do that now.
And we are going to search for SCCM Admin. Click Check Names and we can see that the username is resolved.
You could create a special SCCM client installation user account if you would like, the only thing you need to make sure you do is add it as a member of the Domain Administrators group.
We are just going to use the SCCMAdmin account in this example. Click OK.
Now, you need to type the password for this account. It is really important that you get this password