Immunology of the Female Reproductive Tract

Immunology of the Female ReproductiveTract

Charles R. WiraDepartment of PhysiologyDartmouth Medical School

1 Medical Center DriveLebanon, NH 03756

USA

Mucosal Immunity In The Human FemaleReproductive Tract

• Immune cells in the female reproductive tract.

l Antigen presentation by uterine epithelial cells.

l Localization of HIV receptors and co-receptorsin the uterus.

l Innate Immune responses in the uterus:bactericidal activity and secretory leukocyteprotease inhibitor (SLPI) production byepithelial cells.

Leukocytes in The Human FemaleReproductive Tract

0

10

20

30

% C

D45

Pos

itive

ALL PROLIF. SECRET. INACTIVE

*

*

**

%CD45-POSITIVE CELLSOF TOTAL DISPERSED CELLS

12

0

2

4

6

8

10

Num

ber o

f CD

45 X

106 p

er g

ram

ALL PROLIF. SECRET. INACTIVE

VMECXCXUTFT

*

*

*

NUMBER OF CD45-POSITIVE CELLSPER GRAM OF TISSUE (x 106)

Givan et. al., 1997. Am. J. Reprod. Immunol. 38:350

T Cells (CD3), Neutrophils (Cd66b), B Cells (CD19) and Monocytes(CD14) at Different Stages of the Cycle

FT EM CX ECX VM

B: SECRETORY

CD3

CD66b

CD14

CD190

20

40

60

% O

F C

D45

-PO

SITI

VE C

ELLS

FT UT CX ECX VM

A. PROLIFERATIVE

0

20

40

60

FT UT CX ECX VM

C. INACTIVE

CD3

CD3

CD66b

CD66b

CD14 CD14

CD19 CD19

*

* **

* * *

*

*

*

*

Givan et. al., 1997. Am. J. Reprod. Immunol. 38:350

Mucosal Immunity In The Human FemaleReproductive Tract





Experimental Design: Antigen Presentation,Human Studies

250 µm

40 µm

Enzymaticdigestion ofhuman uterito obtainepithelial &stromal cells

Epithelial cells

3[H]-thymidineProliferation

TT

Purified T cells

Ficoll & T cellenrichmentcolumn

• Confirm PBMCpresentation of TT

• Check T cell purity• Determine if EC

present TT

1. 2.

3.

Antigen Presentation by Mixed Uterine Cellsto Tetanus Toxoid Primed T Cells

#789 #861 #839 #771 #783 #808 #8160

5000

10000

15000

20000

45000

55000

RT+T+TT

RT+T

Proliferative Secretory PostmenopausalMenstrual Cycle (stage)

33 33 H-H- H-H-Th

ymid

ine

Thym

idin

eTh

ymid

ine

Thym

idin

e ( ( ( (cp

mcp

mcp

mcp

m/w

ell)

/wel

l)/w

ell)

/wel

l)

UTERUS

Fahey et. al. Am. J. Reprod. Immunol. 42:49-57,1999

Antigen Presentation By Cervical And VaginalCells To Tetanus Toxoid Primed T Cells

780 771 698 783 8840

500

1000

1500

2000

2500

3000

CX+T+TTCX+T+TTCX+T+TTCX+T+TTCX+TCX+TCX+TCX+T

Menstrual Cycle (stage)Menstrual Cycle (stage)Menstrual Cycle (stage)Menstrual Cycle (stage)

H-t

hym

idin

e (c

pm

/wel

l)3

CERVIXCERVIXCERVIXCERVIX

****

****

latelatelatelateproliferativeproliferativeproliferativeproliferative

earlyearlyearlyearlysecretorysecretorysecretorysecretory secretorysecretorysecretorysecretory postmenopausalpostmenopausalpostmenopausalpostmenopausal

----------------------------------------------------------------------------------------780 771 884

0

300

600

900

1200

1500

VM+T+TTVM+T+TTVM+T+TTVM+T+TTVM+TVM+TVM+TVM+T

Menstrual Cycle (stage)Menstrual Cycle (stage)Menstrual Cycle (stage)Menstrual Cycle (stage)

H-t

hym

idin

e (c

pm

/wel

l)

VAGINAVAGINAVAGINAVAGINA

****

********

latelatelatelate proliferative proliferative proliferative proliferative earlyearlyearlyearly secretory secretory secretory secretory postmenopausalpostmenopausalpostmenopausalpostmenopausal

3

Fahey et. al. Am. J. Reprod. Immunol. 42:49-57,1999

Presentation of Tetanus Toxoid by UterineEpithelial cells

EpithelialCells

T Cells Epithelial& T Cells

0

1000

2000

3000

4000

5000

6000

0 g/ml

10 g/ml

100 g/ml

3 H-T

hym

idin

e (c

pm/w

ell)

Wallace, P.K., Yeaman, G.R., Kristy Johnson, K., Collins, J.E., Guyre, P.M. and Wira, C.R.J.Steroid Biochem. Mol. Biol. . 76(1-5):203-11, 2001

Mucosal Immunity In The HumanFemale Reproductive Tract





Time Course of HIV Infectivity of UterineEpithelial Cells

1 5 15 30 60 120 240 360 7200

5

10

15

20

25

Time of exposure of PHA blasts to HIV-1 (min)

p24

conc

entr

atio

n on

day

10

(ng/

ml)

From: Howell et. al. J. Virol. 71: 3498-3506, 1997.

3 Color Immuno-fluorescent staining.Typically mAbsconjugated to FITC,Cy3 and Cy5

Hysterectomy Tissue.

Prepare vibratome orFrozen Sections.

Fix and mount

Confocal Analysis.Simultaneousacquisition of opticalsections (minimimthickness 0.7µm).

Preparation of Viable Tissue Sections andConfocal Analysis.

1. Uterine epithelial cells express CD4, CCR5 and CXCR4.

• Expression of these receptors varies with the stageof the menstrual cycle.

3. These studies suggest that the uterus may be a site of HIV entry.

Yeaman et al. Unpublished observations

Analysis of CD4, CCR5 and CXCR4CD4, CCR5 and CXCR4CD4, CCR5 and CXCR4CD4, CCR5 and CXCR4 on Viable Tissue Sections by Confocal Analysis

Conclusions

Mucosal Immunity in the Human FemaleReproductive Tract





Effect of Menstrual Cycle Stage and Menopause on Anti-bacterial Activity by Human Uterine Epithelial Cells

ProliferativeProliferativeProliferativeProliferative SecretorySecretorySecretorySecretory InactiveInactiveInactiveInactive0000

20202020

40404040

60606060

80808080

100100100100

120120120120

Menstrual Status

% In

hibi

tion

(+/

- SD)

% In

hibi

tion

(+/

- SD)

% In

hibi

tion

(+/

- SD)

% In

hibi

tion

(+/

- SD)

N=6N=6N=6N=6N=6N=6N=6N=6

N=4N=4N=4N=4

****

Fahey and Wira.. J. Infect. Diseases 185:1606-1613, 2002.

SLPI Concentrations in Apical rinses ofUterine Epithelial Cells

1111 4444 242424240000

4444

8888

12121212

16161616

20202020

24242424

Incubation Time (hrs)Incubation Time (hrs)Incubation Time (hrs)Incubation Time (hrs)

SLPI

(ng

/ml)

SLPI

(ng

/ml)

SLPI

(ng

/ml)

SLPI

(ng

/ml)

Patient 1407Patient 1407Patient 1407Patient 1407


SLPI Concentrations in Water Rinses ofUterine Epithelial Cell Monlayers

1226122612261226 1365136513651365 1407140714071407 1308130813081308 1341134113411341 13971397139713970

3

6

9

12

15

18

21

24

Patient #

SLPI

(ng

/ml)

SLPI

(ng

/ml)

SLPI

(ng

/ml)

SLPI

(ng

/ml)

PPPP LSLSLSLS PPPP IIII IIII IIII

Menstrual statusMenstrual statusMenstrual statusMenstrual status

******** ****


Correlation Between Anti-bacterial Activity and SLPIProduction by Human Uterine Epithelial Cells in Culture

ControlControlControlControl 1111 4444 242424240000

40404040

80808080

120120120120

160160160160

200200200200Colony Forming Units (CFU)SLPI (ng/ml)

Incubation Time (hrs)Incubation Time (hrs)Incubation Time (hrs)Incubation Time (hrs)

Colo

ny F

orm

ing

Units

(CF

U)Co

lony

For

min

g Un

its (

CFU)

Colo

ny F

orm

ing

Units

(CF

U)Co

lony

For

min

g Un

its (

CFU)

SLPI

(ng

/ml)

SLPI

(ng

/ml)

SLPI

(ng

/ml)

SLPI

(ng

/ml)

20202020

16161616

12121212

8888

4444

0000


Neutralization of S. Aureus Anti-Bactericidal Activity inUterine Epithelial Cell Apical Secretionsby anti-human SLPI Antibody

ControlControlControlControl UT SECUT SECUT SECUT SEC UT SECUT SECUT SECUT SEC UT SECUT SECUT SECUT SEC0

20

40

60

80

100

Colo

ny F

orm

ing

Units

(CF

U)Co

lony

For

min

g Un

its (

CFU)

Colo

ny F

orm

ing

Units

(CF

U)Co

lony

For

min

g Un

its (

CFU)

++++ ++++IsotypeIsotypeIsotypeIsotype anti-SLPIanti-SLPIanti-SLPIanti-SLPI

******** ********


ConclusionsConclusionsConclusionsConclusions

• The female reproductive tract (FRT) contains a full spectrum ofimmune cells.

• Antigen presenting cells in the uterus, cervix and vagina are able topresent antigen. Epithelial cells are able to present antigen toautologous T cells.

• Uterine epithelial cells express CD4, CCR5 and CXCR4. Expression ofthese receptors varies with the stage of the menstrual cycle. Thesestudies suggest that the uterus may be a site of HIV entry.

• Uterine epithelial cells produce SLPI, which appears to be underhormonal control, to protect pre-menopausal women from potentialbacterial pathogens.

• Overall, these studies demonstrate that immune cell function in theFRT varies with stage of the menstrual cycle and menopause.

Acknowledgments

Alice GivanAlice GivanAlice GivanAlice GivanPaulPaulPaulPaul Wallace Wallace Wallace Wallace

Grant YeamanGrant YeamanGrant YeamanGrant YeamanJohn FaheyJohn FaheyJohn FaheyJohn FaheyAlix HowellAlix HowellAlix HowellAlix HowellAlice GivanAlice GivanAlice GivanAlice Givan

andandandandGabriellaGabriellaGabriellaGabriella

Dartmouth Medical SchoolLebanon, NH

CLINICAL ACKNOWLEDGMENTS

OR nursesOR nursesOR nursesOR nurses

SchedulingSchedulingSchedulingScheduling

Jeannette Sawyer

Karen Carter

Tracy Stokes

Kris Ramsey

Fran ReinfrankJaclyn Logren

PathologyPathologyPathologyPathology

Clinical Clinical Clinical Clinical SupportSupportSupportSupport

Peter Seery

Tamara Krivi

Maryalice Achbach

Laura Wolfe

Judy Rook

SurgeonsSurgeonsSurgeonsSurgeonsBarry Smith , Chair

Misty Blanchette-PorterJackson BeechamJohn CurrieLeslie Demars

John KettererBenjamin MahlabPaul ManganielloEric Sailer

Susan SteffanJoan BartholdWilliam Young

Paul Hanissian

Immunology of the Female Reproductive Tract

Documents

Transcript of Immunology of the Female Reproductive Tract