# Presentation: 556537

## MK-571, an inhibitor of Multidrug Resistant Protein 1 (MRP1) selectively blocks T cell activation and may reveal novel drug targets for immunomodulation

**Ronald L. Rabin, M.D.**
**Laboratory of Immunobiochemistry**
**                                                              ****rabin@cber.fda.gov**

## observation

**Activation affects [probe]                 [probe] reflects gene expression of probe transporter                                 Does probe transporter modulate activation? **

**Notes:**
this is a cartoon of the observation.  Less probe in WBC from patients with Wegener’s Granulomatosis.

## MDR Family

**Proteins that transport substances across cellular membranes, against a concentration gradient, in an energy dependent manner.**
**ABC proteins (*****A*****TP *****B*****inding *****C*****assette) that contain distinctive nucleotide binding domains (NBD).**
**Genes are highly conserved across species.**
**First member is MDR1 (P-glycoprotein, P-gp); best substrates are large hydrophobic cations:**
**	****Doxorubicin**
**	****Ritonavir**
**	****Calcein-AM**
**	****Verapamil**
**MDR-associated Resistant Protein-1 (MRP1) described in 1992; 15% homologous to MDR, more closely related to CFTR:**
**	****organic anions**
**	****glutathione, glucuronide, & sulfate conjugates**
**	****LTC****4**
**	****Calcein****	**

## MDR Family members

**MDR Family members**

**Percent identity between MDR members**

## MDR family gene expression in T cells35 cycle RT-PCR specific products

**ACTIN**

**B cells**

**NK cells**

**Monocytes**

**0 day**

**3 days**

**memory**

**naive**

**memory**

**naive**

**MRP4**

**CD4**

**CD8**

**Cord CD4**

**Notes:**
Some of the members are expressed in memory not naïve cells.  The cord “3 day” is 3 days after stimulation with anti-CD3 and anti-CD28.

## MK-571 is an inhibitor of MRP1

**MK-571 is an inhibitor of MRP1**

**TSST-1 10  ng/ ml**

**Control**

**40x**

**MK-571 (100μM)**

## The MRP1 inhibitor MK-571 decreases expression of CD69 in response to superantigens

## The MRP1 inhibitor MK-571 decreases IFN- but not IL-4 TSST-1 stimulated human CD4 T cells

## The MRP1 inhibitor MK-571 blocks IFN- synthesis

## The MRP1 inhibitor MK-571 blocks cytokine secretion

**The MRP1 inhibitor MK-571 blocks cytokine secretion**

## The MRP1 inhibitor MK-571 blocks TNF- secretion by human macrophages

**The MRP1 inhibitor MK-571 blocks TNF-******** secretion by human macrophages**

**MK-571 (μM)**

**TNF-******** (pg/ml x 10****3****)**
**in response to SAC**

**TNF-******** (pg/ml x 10****3****)**
**in response to LPS**

**Notes:**
Three different donors

## The MRP1 inhibitor MK-571 does not block IL-6 secretion by human macrophages

**The MRP1 inhibitor MK-571 does not block IL-6 secretion by human macrophages**

**MK-571 (μM)**

**Notes:**
Three different donors, same sups as TNF

## The MRP1 inhibitor MK-571 blocks calcium flux in response to T cell receptor cross-linking

**The MRP1 inhibitor MK-571 blocks calcium flux in response to T cell receptor cross-linking**

**Ratio fluorescence (violet/blue)**

**Relative number of cells**

**Notes:**
This was done twice, but only once with 5-LO pathway inhibitors

## The MRP1 inhibitor MK-571 blocks calcium flux in response to the chemokine SDF-1 (CXCL12)

**Notes:**
Histograms from 11/6; CXCR4 staining was done by me and done at least 2 or three times.  Calcium done twice.

## MRP1 blockade is reversible

**MRP1 blockade is reversible**

**Percent Inhibition**

**Percent IFN-********+**
**CD4 T cells**

## The MRP1 inhibitor MK-571 is ineffective if added two hours after T cell stimulation

**Percent IFN-********+**
**CD4 T cells **

## Is MRP4 the Th2 counterpart to MRP1?

**•****MRP4 transports “the other” arachidonic acid products, PGA****2****, PGE****1****, PGE****2****, and PGF****1a**** and PGF****2a****, and PGD****2****. **
**•****PGE****1**** down-modulates Th1 activity and up-modulates Th2 activity**
**•****The PGD****2**** receptor, CRTH2 is used to define Th2 cells**
**•****Do Th2 cell lines express more MRP4 than Th1 lines?**
** **

***Reid et al.  PNAS 100:9244; 2003***

## Increased expression of MRP4 in Th2 cell lines

## RSV may increase expression of MRP4 in Human Monocyte Derived Dendritic Cells

**RSV may increase expression of MRP4 in Human Monocyte Derived Dendritic Cells**

## Conclusions

**MRP1 blockade abrogates:**
**	****type 1 (e.g. IFN-********) T cell function**
**	****calcium flux after TcR stimulation**
**	****calcium flux after chemokine receptor stimulation**
**MRP4 may regulate type 2 T cell responses analogous to MRP1/Type 1.**
**Defining the mechanisms by which MRP1 and MRP4 regulate T cell function may provide novel drug targets for immunomodulation.**

## Acknowledgments