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CYTOKINE EXPRESSION DURING DIFFERENT PHASES OF THE MENSTRUAL CYCLE
by Christine April King
Athesissubmitted to the
School of Graduate Studies inpartialfulfilment of the requirements for the degree of
Master of Science
Faculty of Medicine MemorialUniversity ofNewfoundland
March 1998
St.John's Newfoundland
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ABSTRACT
Previous studieshave shown that cultured and circulating cells from women may differ inimmune responses, the differences relating to the phase of the menstrual cycleinwhich they were harvested.Inthisinvestigation, specific enzyme-linked immunosorbent assays forIL-2. IL--4,IFNY,IL-Ira andn.~lllwereusedto determineifthere are differencesin levels of cytokine expression at different phases of the menstrual cycle.
A sample set of mononuclear cells and serum were isolated fromnine healthy.
cycling females(19-29years) at known pointsintheir cycle and from four male controls. The gonadal hormones 171J-estradiol, progesterone and luteinizing hormone. as well as serum cytokine levels and cytokineproductionby unstimulated and stimulated cellsin culture were monitored Results indicated that the levelsof all the cytckmes measured varied in relation to the menstrual cycle. lL·2.[L-4,and lFNy secretion byPHA stimulated PBMCwas low at time of ovulation and highinthe luteal phaseof the menstrual cycle.There appearstobe a TKI to TR2 shift from the follicular to theluteal phase. Secretion of IL-lpand Ils-Ira in LPS stimulated cultures changed during the menstrual cycle. The IL·I ratio (ll.-I fl/ll..-l fa)waslow at ovulationin unstimulated cultures and low at the late follicular and the midluteal phases of the cycle in LPS stimulated cultures. The IL-Iratioinserum was lowest at ovulation with higher levels inthe follicular and luteal phases. Variabilityinthe levels of the cytokines detectedin successivesamples.both in serum. andinculture supernatants. was greater for women than
men for IL-4,lFNy in culture supernatants,and for lL-lra and IL-II}in serum. In addition. females had significantlyhigher mean values for IL-Ira in nonstimulated cultures and lower mean [L.I ratios in serum as comparedtomales. Finally, multiple regression analysisof the cytokine datawiththe hormones estrogen, progesterone. and luteinizing hormone as well as with age of the volunteer,dayinthe cycle the sample was obtained, length of cycle. and day of ovulation demonstrated that 1L-4 and possibly IFNy production by stimulatedPBMC and IL-Ira production by unstimulatedPBMeare positivelycorrelated with age. Incontrast,1L-2, productionbystimulatedPBMCand serum levels of IL-Ira and IL-IJJ are negatively correlated with age. D..-Ira secreted by both stimulated and nonstimulated cells ispositivelycorrelated with day in the cycle the sample is taken whereas IL-I
P
correlateswithday ofovulation and length of cycle. All of thecorrelations that were found to be significant in this study suggest thar the menstrual cycle has a marked effect on the levels ofeytokine production as detected in vivoinserum samples and invitrointhe supernatants of cellsin culture.Insummary,these data demonstrate that a) thereis greater variation over time in amounts of cytckine produced by women than by men b) there are changesin cytokine production in women related 10 the menstrual cycle c) the lowest eytokine production occurs at time of ovulation (the exception being IL-I in stimulated cultures) and d) age influences the amounts of cytokines producedbywomen.
The data presented heresupport the hypothesis that (i) there arecyclicalchanges inimmune responses in women (ii)theimm uneresponseis influencedbychangesin
ii
hormonestatusand(iii) regulationofcytokineprodu ctionis fundamentally differentin women than men.
iii
ACKNOWLEDGEMENTS
Iwish tosaya heartfeltthankyoutomysupervisor, Dr.Bodd Larsen.who has been therewithsuppo rt,encouragement and sound advicethroughout these pastyean.
A specialthankyo u to the other members of mysupervisory committee.Dr.Vema Skanesand Dr.Ehud Ur,who provided mewithuseful advice about myresearch andthis manuscript
To Dr.VeerabhadtaGadeg,who helped medecipher the enormous amount ofdata I had collected and to moulditintosomething understandable,thankyou for all the help yougave meon thestatisticalaspectsof my analysis.
To IU my volunteers.boththe ladies who so graciously gave an enormous amount of their time and energy.as well as blood. for many months dwing thisstudyand to the gendeman who reliablyturnedupthree times per weekand donated blood. Ithank:you for your cooperation;withoutyou I would not have been able to do this project.
To Carolyn Murphy,thank you for helpingin obtaining the blood samples needed fromallthe volunteers in thestudy.
To everyone in Immunology,thank you for helping me whenever you could and for never tiring of my seemingly endless questions.
To myparents,Dianne and Wayne King, for providing mewithall the support I neededto continue on this paththatI have chosen,a loving thank you.
And finally,10Stephen Hennessey,a sincere and heartfelt thank you for being
iv
there through these last three years, for putting upwithmy worries about my research and for listening to me talk about immunology every day of mylife.
Financial supportwasprovidedbyDr.Bodil Larsen and the Faculty of Medicine.
TABLE OF CONTENTS
Abstract Acknowledgements list ofTables Listof Figures List of Abbreviations
Chapter [ - Introduction.
1.1The Menstrual Cycle
1.1.1The feedback Relationshipbetween Ovarian and Pituitary Hormones
1.1 .2Beginning of the Cycle 1.1.3Follicular Phase 1.1.4Ovulation . 1.1.5The Luteal Phase 1.2Sex Hormones and Immune Response
1.2.1Sex Related Differences in Immune Response 1.2.2HumoralImmunity
1.2.2.1 DirectAction of Gonadal Hormones
vi
iv ..xiv ..xviii xxi
1.2.2.2Indirect Action of Gonadal Honnones 1.2.3 Cellular Immunity.
1.2.4The Cycle and Immune Response. 1.2.4.Humoral and Mucosal Immunity 1.2.4.2 Prostaglandins
1.2.4.3Cellular Immunity 1.2.5 Conclusions
1.3Cytokines 1.3.1InterleukinI. 1.3.2Tumour Necrosis Factora 1.3.3Interleukin 6. 1.3.4lnterleukin 2. 1.3.5Interleukin4. 1.3.6Interferon'Y 1.4 Sex Hormones and Cytokines
1.4.1Sex Hormones and IL-l . 1.4.2Sex Hormones and TNFa 1.4.3Sex Hormones and Other Cytokines 1.4.4Pilot Study.
1.4.5Conclusions 1.5 Aims and Objectives
vii
12 12 13
13 16 17 20
21 21 23 24 25 26 27 28
28 34 35 37 38 43
Chapter D - Materials and Methods 2.1Volunteers
2.1.1Cycling Women selection Process 2.1.2Control Selection Process 2.2Phase I
2.2.1Menstrual Cycle Diary. 2.2.2OvuQuick Kit 2.3Phase
n
2.3 .1Collection of Blood Samples 2.3.1.1Preparation of Cells 2.3.1.2Culture Medium. 2.3.2Stimulation Protocol
2.3.2.tInduction of lL·2 and [L-4 Secretion 2.3.2.2Induction of IL-H3 and [L-Ira Secretion..
2.3.2.3Induction ofIFN-ySecretion 2.3.2.4Harvest of Culture Supernatants. 2.3 .3Serum Samples...
2.4ELISA Assay s.
2.4.1General Principleof the [L-2 Immunoassay. 2.4.1.1Procedure
2.4.2General Principle of theIT..-4Immunoassay(I)
viii
45 45 45 45 46 47 47 48 48 49 50 50 50 51
51 51 51 52 52 54 55
2.4.2.tProcedure
2.4.3General Principle of the1l.-4 Immunoassaytlf ] 2.4.3.1Procedure ...
2.4.4GeneralPrincipleof thelL·lraImmunoassay 2.4.4.1Procedure....
2.4.5 General Principleof thelL-IIlUltrasensitiveImmuncassey 2.4.5.1Procedure
2.4.6GeneralPrinciple of the
n...lp
Immunoassay 2.4.6.1Procedure2.4.7GeneralPrinciple of theIFN-yImmunoassay.
2.4.7.1Procedure .
2.4.8GeneralPrinciplesofthefi...6 Ultraseositive Immunoassay 2.4.8.1Procedure
2.4.9GeneralPrinciplesoftheTNF-o.UltrasensitivcImmunoassay
2.4.9.1Procedure .
2.5Hormone Radioimmunoassays..
2.5.1GeneralPrincipleofthe17Jl-Estrawol RIA 2.51.1RIA Protocol
2.5.2GeneralPrincipleoftheProgesteroneRIA
2.5.2.1 RIA Protocol .
2.5.3GeneralPrincipleof theTestosteroneRIA.. . . .
ix
55 56 57 58 59 60 61 62 62 64 64 65 66 67 67 68 70 70 72 72 73
2.5.3.1RIA Protocol.
2.5.4General Principle of the Luteinizing Hormone RIA 2.5.4.1RIA Protocol .
2.6C'H50and C3
2.7Coefficient of Variation (CV) 2.8Statistical Methods
2.8.1Coefficient of Variation. 2.8.2F Test
2.8.3tTest. 2.8.4Multiple Regression
Chapterill~Results
3.1Establishment of SimulationConditions 3.2Creation of a Sample Set fromFemaleVolunteers
3.2.1Sampling Days
3.3Creation of a Sample Set from Male Controls.. 3.4HormoneProfiles. . .
3.4.1GroupMeans 3.4.2Coefficient of Variation. 3.4.3Summary..
3.5Variability between Cycling Females and Males
73 74
74 75 76 76 76 77 77 78
80 80 88 90 92
94 104 106 106 108
3.5.1Coefficient of Variation. 108
3.5.2Precision in Cell Counts 110
3.5.3The F Test . 110
3.5 .4Comparison of Means between CyclingFemales and Males. 112 3.6Cytokine Levels During the MenstrualCycle 112
3.7Cyclical VariationinFemales 115
3.7 .1Proportionsof the MenstrualCycle. 116
3.7 .2CompositeHormone Profile. . 118
3.7.3CytokineAnalysis. 118
3.7.3.1lnterleukin2 121
3.7.3.2Interleukin 4 121
3.7.3.3Interferony. 123
3.7.3.4lnterleukin Irain nonstimulated cultures 126 3.7.3.5lnterleukinIra instimulated cultures 126 3.7.3.6Interleukin
Ip
in nonstimulated cultures 128 3.7.3.7lnterleukinIP
in stimulated cultures 1283.7.3.8Interleukin I 130
3.7.3.9ProblemswithIl>!Data 130
3.7.3.10Serum lnterleukin Ira 132
3.7.3.11Serum lnterleukin
IP
1323.7.3.12Serumn-iRatio. 135
3.7.3.13IL-6andTNFa 3.7.4Summary
3.8Regressionsand Correlations . 3.8.11L-2.
3.8.21L-4. 3.8.3lFNy 3.8.4Culture IL-I ra 3.8.5Culture1L-1P 3.8.6Serum1L-1
3.8.7Serum LevelsandNonstimulated Productionofn..-I
3.8.8Summary .
3.9JustificationofStatisticalMethods .
3.10Technical Problems .
Chapter IV - Discussion 4.1Creationof Sample Set.
4.1.1FemaleSampleSet
4.1.2MaleControl SampleSet 4.1.3Hormone Level s.
4.2VariabilitybetweenCyclingFemalesand Males 4.2.11L-2,1L-4andlFNy
xii
135 135
138 138
140 142 142 146 149 152 154 154 157
160
160 160 163 164 165 166
4.2.2Culture IL-I 4.2.3SerumIL·I
4.3Cytokines and The Menstrual Cycle. 4.3.11L-2,1L-4and IFNy 4.3.2Culture IL·I . 4.3.3SerumIL-I 4.4 Correlations and Regressions
4.4.11L-2,1L-4and IFNy 4.4.2Culture IL·I 4.4.3Serum IL·I 4.5C3 and C'H50 4.6Receptors 4.7Conclusions. 4.8 Future Studies.
References
Appendix A Appendix B Appendix C
xiii
166 169
169 170 174 176 178 179 179 181 182 18J 187 189
191
202 212 248
l.Table1.1:
2.TableI.2:
3.Table 2.1:
4.Table 2.2:
LIST OF TABLES
Plasma concentrations.production rates,and ovariansecretion rates
of ovarian steroidhormones. 4
Summary of bumancytokine datain relation to the female gonadal
hormones. 40
Summary of the cytokines examinedfor eachafthe volunteerswith reference tothe commercial source oftheELISA,sample and samplesize and sensitivityof theassay. 53 Swnmaryof thehormonesmeasuredforeach afthe volunteerswith reference to source of theRIA,sample andsample size ,and
sensiti vity of the assay. 69
5.Table 3.la: Time study measuringincorporation oflH-thymidine. 82 6.Table 3.lb:
7.Table 3.lc :
8.Table 3.ld:
9.Table 3.2:
10.Table 3.3:
II. Table 3.4:
12.Table3.5 :
Timestudy measuring incorporation ofIH· thymidine at25~g/ml
PHA. 82
Time studymeasuring incorporationof lH.thymidineat 50J.lglml
PHA. ~
Time study measuringincorpo ration oflH-thymidine at75J.lg/ml
PHA 83
Cyto kine concentrationsinculture supernatants using various
stimulation conditionsfor24 hours 8S
Cytokinesin culture supernatantsafter stimulation of PBMCwith three differentconcentrations of PHA for 24 hoursin media
supplementedwith1%FCS. 87
lFNy concentrations in culture supe rnatants when PBMC are stimulated for 48 and 72hours with two different concentrations of PHAinmedia supplementedwith1%FCS.. . 87 Ovulation data and length of cycle of female volunteers
participatinginPhase [ 89
xiv
13.Table 3.6: Cycledataduring Phase
n
for 9 females. 91 14.Tabl. 3.7 : Expected and actual ranges,and group means,for each of the gonadal hormones measured in this study. ... lOS 15.Tabl. 3.8: Coefficientsofvariation for the RIA assays 107 16.Tabl. 3.9 :17.Tabl. 3.10 : 18.Tabl. 3.11:
19.Tabl. 3.12:
20.Tabl. 3.13 :
21. Tabl. 3.14:
22.Tabl.3.15:
23.Tabl. 3.16:
24.Tabl. 3.17 :
25.Tabl. 3.18 :
26.Tabl. 3.19:
27.Tabl.3.20:
Coefficients of variation for the EUSA assays 109 Ftestdataon each of the cytokinesexaminedinthestu dy 111 The means,standard error.ranges and p values for males and females cytokinesassayedfrom culture supernatants. 113 The means, standard error,ranges and pvalues for each group for the cytolcinell.·1assayedin serum from males and females .114 Asummary of the changesinrelative levels of cytokines that were foundtooccur during the course of the menstrual cycle .. 137 Variablesinthe predictor equation for [L·2withthe 95% confidence-
intervals and thepvalues... . ... . .. 139
Variablesinthe predictor equation for 1L-4withthe 95% confidence
intervals and thepvalues. 141
Variables in the predictor equation forlNFy with the 95%
confidenceintervalsand thepvalues.. 143 Variables in the predictor equation for lL-lrainnonstimulated cultureswiththe 95% confidence intervals and the p values. 144 Variables in the predictor equation for !Lei rain stimulated cultures with the 95% confidence intervals and the p values. 145 Variablesin the predictor equation for ll.-IP in nonstimulated cultureswiththe 95% confidence intervals and the p values. 147 Variablesinthe predictor equation for ILelPin stimulated cultures withthe 95% confidence intervals and the p values. 148
28.Table 3.21:
29.Table 3.22:
30.Table 3.23:
28.Table A.I :
Variablesinthe predictor equation for IL-Ira in serum samples with the 95% confidence intervals and thepvalues. 150 Variablesinthe predictor equation for IL-I~inserum sampleswith the95%confidence intervals and thepvalues. 151 Variables, determinedby multiple regression,that significantly influence, positively(+)or negatively (-),the cytokines measured
in females 155
The volunteers hemoglobin levels and various cell population numbers(109/L )3tthe beginning of thestudy(8)and at the end
(E). 211
29.Table 8.1-B.15 :Original sample data. means, and standard deviations, for all the
volunteers in thestudy. 213
30.Table 8.16-8.30:Original sample data, means and standard deviations,for all the
volunteersinthestudy. 229
31. TableD.31 :
32.Table D.32:
33.Table C.I:
34.Table C.2:
35. Table C.3:
36.Table C.4:
37.Table C.5:
Means and standard deviations for the phase of the cycle profiles for all the cytokines measured in culture supernatants. 245 Means and standard deviations for the phase of the cycle profiles for all the immune parameters measured in serum. 247 Values for the same serum sample run on different RIA estrogen assays with the calculated coefficient of variation. 250 Values for the same serum sample run on different RIA progesterone assays with the calculated coefficient of variation. 252 Values for the same serum sample run on different RIA luteinizing hormone assayswiththe calculated coefficient of variation 254 Values for the same serum sample run on different RIA testosterone assays with the calculated coefficient of variation. 256 Values for the same culture supernatant sample run on different Interleukin 2 ELISA assays with the calculated coefficient of
variation. 258
38.Table C.6:
39.Table C.7:
40.Table C.8:
41. Table C.9:
42.Table C.IO:
43.Table C.1I :
Values for the same culture supernatantsamplerun on different lnterleukin 4 ELISA assays with the calculated coefficientof variation. . .. .... . ... ... ... . . . .. 261 Valuesfor the same culture supernatant samplerun on different Interferon gamma EUSA assayswiththe calculated coefficient of
variation... .. . . ... 263
Values for the same culture supernatant sample run on different lnterleukinIra EUSA assayswiththecalculated coefficientof
vari ation. 265
Values for the same serum sample run on different lnterleukin Ifa ELISAassays with the calculated coefficientof variation. 267 Values for the same culturesupernatant sample run on different lnterleukin1~ELISAassayswiththe calculated coefficientof
variation. . . ... 269
Valuesfor the same serum sample run on different Interleukin lP EUSA assayswiththecalculatedcoefficient of variation... 271
xvii
I.Figure1.1:
2.Figure3.1 :
3.Figure 3.2; 4.Figure3.3: 6.Figure3.4: 7.Figure3.5: 8.Figure3.6: 9.Figure3.7 : 10.Figure 3.8; II. Figure3.9: 12 Figure3.10 ; 13.Figure 3.11 ;
14.Figure3.12:
15.Figure 3.13;
16.Figure3.14:
17.Figure 3.15;
LIST OF FIGURES
Relative levels of estradiol. progesterone, FSH and LH throughout
the menstrual cycle. 3
Day of sampling for each of the nine volunteers expressed as
proportion of phase. 93
Hormone profile of volunteer21.04.70. 95
Hormone profile of volunteer01.04.75 96
Hormone profile of volunteer22.11.75 97
Hormone profile of volunteer15.12 .73 98
Hormone profile of volunteer29.08.71 99
Hormone profile of volunteer22.09 .75 100
Hormone profile ofvohmteer06.09 .72 101
Hormone profile of volunteer09.10.64 102
Hormone profile of volunteer30.04.65 103 Composite hormone profile as seen when the cycle is converted into
proportion of phase. 119
The1L-2cytokine profile in relation to phases of the menstrual
cycle. 122
The1L.-4 cytokineprofile in relation to phases of the menstrual cycle. ... . .. . . ... 124 The lNFy eytokine profileinrelation to phases of the menstrual
cycle. 125
TheIL~1ra cytokine profile in relation to phases of the menstrual cycleinncnstimulated and stimulated cultures. 127
xviii
18. Figure 3.16:
19.Figure 3.17 :
20.Figure 3.18:
21. Figure 3.19 :
22.Figure 3.20:
23.Figure C.I:
24.Figure C2:
25. Figure C.3 ;
26. Figuree.4:
27.FigureC.S:
28.Figure C.6:
29.Figure C.7:
30.Figuree.8:
31. Figure C.9 :
The IT..-I
P
cytokine profile in relation to phases of the menstrual cycleinnonstimulated and stimulated cultures. 129 TheIls-1ratio cytokine profileinrelation to phases of the menstrualcycle. 131
Serum IL·I ra cytokine profile in relation to phases of the menstrual
cycleinserum.. 133
Serum IL-Ill cytokineprofile in relation to phases of the menstrual
cycleinserum 134
The IL-lratio cytokine profile in relationtophases of the menstrual
cycle. 136
Standard curves from the Estradiol RIA run at four different
times. 249
Standard curves from the Progesterone RlA run at four different
times. 251
Standard curves from the Luteinizing hormone RIA run at four
different times. 253
Standard curves from the Testosterone RIA run at three different
times. 255
Standard curves from the IL-2 EUSA run at six different
times. 257
Standard curve from the R&D Systems 1L-4 ELISA run at three
different times.. 259
Standard curves from the Biosource[L.4EUSA run at five different
times. 260
Standard curves from the IFNy EUSA run at eight different
times. 262
Standard curves from the IL-Ira ELISA run at ten different times to measurell.-1ra in culture supernatant. 264
xix
32.Figure C.1O:
33.Figure C.ll :
34.Figure C.12:
Standard curves from the IL- Ira ELISAnut at five different
times tomeasure IL-I ra in serum. 266
Standard curvesfrom the IL·{
P
EliSA run at ten different timesto measure IL-IJl in culture supernatants. 268 Standard curves from the IL·IJlEUSA run at five different timesto measure n..·IJlinserum. 270
xx
Ab ACO Ag CPM OHEA OHT E2 EBY EF EL ELISA FeR FCS FSH GM·CSF GnRH HLA IFNy Ig IgA IgE IgG
n-t
ll.-I~
n..·lra ll.-2 ll.-4
u.s
i.m i.n.
i.p.
K"
L LF LH LL LPS M M
LIST OF ABBREVIATIONS
Antibody Acid Citrate Dextrose Antigen
Counts per minute Dehydroepiandrosterone 5a.-di-bydrotestosterone 17~Estradiol Epstein Barr Virus Early Follicular phase Early Luteal phase
Enzyme Linked Immunosorbent Assay Fe receptor
Foetal Calf Serum Follicle Stimulating Hormone
Granulocyte Monocyte Colony Stimulating Factor Gonadotropin Releasing Hormone
Human Leukocyte Antigen Interferon gamma Immunoglobulin Immunoglobulin A Immunoglobulin E lmmunoglobulin G lntetleukin1 lnterleukin1beta
lnterleukin1receptor antagonist Inrerleukin2
Interleukin4 Interleukin 6 Intramuscular Intranasal Intraperitoneal Disassociation Constant Litre
Late Follicular Pbase Luteinizing Hormone Late Luteal Phase Lipopolysaccharide Menses Molar
xxi
MCP-l MF MIlC ML
ml
MLR mM mRNA mg ng NK
o
00 OS P PAC PBL PBMC PFC PG pg PHA PMA PWM RIA SC s.c.
SO SRBC To THI TH2 TNFex TNFR TNFRI TNFRll U
~g
~I VCAM·I vs
Monocyte ChemotacticProtein-I MidfollicularPhase
MajorHistocompatibilityComplex Midluteal Phase
Millilitres
Mixed Lymphocyte Reaction Millimolar
Messenger Ribonucleic Acid Milligrams
Nanograms Natural Killer Cells Nanometers Ovulation Optical Density Donor'sown serum Progesterone Peritoneal Adherent Cells Peripheral Blood Lymphocyte Peripheral Blood Mononuclear Cells Plaque Forming Cells
Prostaglandins Picograms Phytohaemagglutinin Phorbal 12-myristateta-acerate Pokeweed mitogen Radioimmunoassay Secretory Component Subcutaneous Standard Deviation Sheep Rod Blood Cells Testosterone T HelperDoe T Helper two
Tumor NecrosisFactor alpha Tumor Necrosis Factor Receptors TNF Receptor Type I TNF Receptor Type D Units
Micrograms Microlitres
Vascular Cell Adhesion Molecule 1 Versus
xxii
CHAPTER I INTRODUcnON
Itis generally recognized that women havemore vigourousim mune responsesthan men. lt would appear that gender influencesbothhumoral and cell mediatedim m un e responses sinc ea number of autoimmune diseasesoccurwith higher frequencyin women thanin men.
Itis suggested that a woman's imm une responses are influencedby hormones,and, sincehormone levels change throughout the menstrual cycle one would expect her immune responses to also change.
I have followed nine women through a full menstrual cycle.Hormone levels and cytckines were measuredin blood samples taken in different phasesof the cycle.The aim of the study was toseeifchanges in cytokine production correlatedwiththe phases of the menstrual cycle.
1.1 The Menstrual Cycle
Females undergo complexcycling of reproductive hormones;this cyclin g is referred to as the menstrual cycle (or perhaps more correctly,the ovariancycle ).
The menstrual cycle alternates between two major phases,thefollicular phase, which typicallypersistfor12 to 16 days and is characterizedbythe presence of maturing follicles, and the luteal phase, which most commonlypersistsfor 10 to 16 days and is charscterisedby the prese- e of tit,.corpus luteumin the ovary (Marshall,1995).
The cycling that isexhibitedin femalesis due tothe ovarian and pituitary hormonesthat are produced. Thelevels of thesehormon esthatare seenin a typical femalecycle are illustr atedin Figure1.1(modifi edfrom Assc,198 3)and theplasma concentration ofvarious ovariansteroids as wellas theirproduction andsecreti onrates arc showninTable1.1(modified from Hsuch et a/., 1995).
1.1.1 The Feedback Relationship between Ovariu and Pituitary Hormones The feedb ack relationship that exists between the ovarianhormone estrogen and thepituitaryhormon es.folliclestimulating hormone(FSH) andluteinizing hormone(LH), determines the events of themenstrualcycle (Asso, 1983). Themainestrogenicprodu ct of the ovaryis17~estradiol(E2)andit also producesprogesterone(P).FSH and LH are controlledby the hypothalamus throughgonado tropin releasinghormone(GnRH).
The contr ol mechanismsarea combination of negativeand positive feedback systems (Clarke,199 5). Estrogen exertsnegative feedbackcontrol on FSHand LH throughGnRH.Ifestrogenis increased,the tendency istosuppressGnRH.releaseinthe hypothalamus.thusintumsuppressing FSH andLH productionandrelease.Inaddition, theovarianhormonesalso act atthepituitaryleveltodecreasethesensitivity of FSH and LH producing cells toGnRH(Asso,1983). However ,there seemstobe a uniqueswitch in the effectofestrog en atthetime ofthepreovulatoryGnRHILHsurge.At this timethe situationisreversed andestrogenexertsapositive feedback:effecton the system(Clarke, 1995).
~ c:
c:uo c:o tJ ec:
eE :::Q
Ui
,.-
/ Oestradiol,'
~'
V
---
• Menses
~Ovulation ,\rogesterone
Figure 1.1. Relative levels of estradiol, progesterone.FSH and LH throughout the menstrualcycle.
Table 1.1.Plasmaconcentrations,production rates, and ovariansecretion ratesof ovarian steroidhormones.
Steroid Pbase of Plasma cone. Production Secretionrate Menstrual <ng/IOOmI) rare (mglday) (mglda y) Cycle
Estradinl Earlyfollicular 008 0.07
Late follicular 33-70 0.5-1.0 0.4-0.8
Midluteal 20 0.27 0.25
Estrone Earlyfollicular 0.11 0.08
Late follicular 15- 30 0.3-0.7 0.25-0.50
Midluteal II 0.24 0.16
Progesterone Follicular 50-100 2.1 1.5
Luteal 1000-1500 25.0 24.0
17a- Early follicular 30 0.6 0.2
hydroxyproges terone
Late follicular 200 4.0 3-4
MidluteaJ 200 4.0 3-4
Androsten- 130-160 3.2 0.8-1.6
edione
Testosterone 38 0.3
1.1.2 BqiDOiDZ:or the Cycle
The beginning of thecycleis referred to as Day I and is thefirstday of men.struation.Thelevels of hormones at the end of one cycle are preparing thebodyfor the next cycle.Thiscanbeseenin the honnone FSH. Asestrogen levelsdeclinetoward thelatelutealpbase,FSH is beginning to risecommencing the growth of folliclesthat willbe involvedinthenext cycle(Asso. 1983).
1.1.3 Follic ular Phase
The follicularphase begins withDayIof the cycle andendswithovulation.
Throughout this phase thefollicles that caneventual ly give riseto a matureovum influence thechanges seeninthisphas eandthe hormonalchangesthatoccurin this phase providethe correct environmentfor maturation of the follicl e.
FSH stimulatesthegrowthof thesefollicleswhich intumbecome thesource of estrogeninthe cycle(Asso , 1983). Thusthe earlyfollicul ar phaseischaracterizedby relativelyhigh plasma levelsof FSH and10wlevelsofLH..estrogen,progesterone,and inh ibin. FSHandLHexert a combine deffect on the folliclestostimulatesecretion of estradiolwhichbythe middletothe late follicularphase is secrete dby thedominant follicle (Marshall,1995).
Inthemidfollicularphase estradiol concentrations in plasma are risingand suppressingsecreti on of FSHbyactionODthe pituitary.Aswell.lohibinlevels are rising possiblycontribu tingto thesuppressionof FSH (Marshall.l99S ).
Esuadiol is increasedinthe late follicular phase and exerts a positivefeedb ack effect on LHby enhancing LHrespo esrveness eoGnRHand by in creasingGn.RH secretion (Marshall. 1995).
Progesterone.at this timeinthe cycle.is also beginningtoriseand exerts a positive feedback effect on LH. lt is the"o varian estradiol-progesteronesignal system"
thatinduces the"GnRH -LH· FSH" ovulatory surge (Marshall. 1995).
Several follicles developinthe ovary at the same time but only onewill go on to become the ovum thatisreleasedat ovulation. Theother follicles will undergoatresia, The developing or atreticstage of a follicle is characterizedby the follicular fluid content of estrogensand androgens (Hsueheral.,199 5).Asthey undergo atresia the follicleswill beginco switch fromsecreting principally estrogen to the secretion of'androgens.One of theandrogens thatis secretedinthis stage is testosteroneand itis seen to peak just prior to ovulation(Asso .1983)
The menstrual cycle of' a woman is generallystated to be 28 daysinlength.
However,the length variesgreatly between women. On averagethe cycleis 29.5days witha range of 26·34days in normal females (Asso,1983).
1.1.4 Ovu•• rioa
As estrogen declines in the late follicular phase a surgeinLH is seen and maintained for approximately24 hours. This UI surge causes the largest follicle of the ovumto be released from the ovary thereby allowing for possiblefertilizationand
6
implantation(Asso,1983).
The rupture of the follicle at ovulation signals the end of the follicular phase and the beginning of the luteal pbase (Marshall. 1995).
1.15 TheLuteal Ph ...
The ruptured follicle that is left bebind in the ovary following release of the oocyte undergoes a process called luteinization resultinginthe formation of the corpus luteum (Marshall,1995).
This corpus luteum is responsible for the production and secretion ofprogesterone and estrogen that are seeninthe luteal phase of the menstrual cycle.
This increased secretion of progesterone and estrogen lasts for a number of days after ovulation during which time FSH secretion is inhibited andremains low (Marshall.
1995).
This increase in progesterone stimulates the growth of the endometrium that will allow for development and growth of the fertilized ovum (Asso,1983). If fertilization does Dot take place.plasma levels of estradiol and progesteronedecline leading to shedding of the endometrium and is seen as menstrual flow(Asso, 1983).
At this time FSH levels begin to riseinitiating the developmentofanew set of follicles,beginning the cycle alloveragain (Asso, 1983).
1.2SeE.bormoDes and Immune Response
Ithas long been known that the sex hormonesinfl uence or are interrelated with the immuneresponse.Researchin the field ofimmune-endocrine interactions has been ongoingfor some time. Four main areas of researchtoday haveprovided significant evidence forthe role of sex hormonesin theimmune response, including: I) the differencesinimmune responses between males and females;2)the knowledge that gonadectomy and hormone replacementalterthe immune response;3)the observationthat theimmune responseis altered during pregnancy;and4)the presenceof receptorsfor sex hormonesoncelts of various arms of theimmune system (Grossman,1984).
1.2.1 Sex Related Differences in Immune Response
Sex related differencesinimmune response have been observedinseveral strains ofmice andin humans (Huber et aJ.,1981;Shuurs and Verheul,1990). It has been observed that males are less likely to develop autoimmunediseases than females (Lillehoj etal.,1981;Ahmed and Penhale,1982),that autoimmunityinmalescan be induced by injection of estradiol(Gersh win et al.,1980)and that femaleshave highertitres of autoantibodiesin serum (Inman, 1978;Ahmed etat.,1985). Theimmune responseof females is greater than that of males (Grossman,1985). Females havehigher serum immunoglobulin levels than males,mounthigher antibody (Ab) responses to various antigens(Schuurs and Verheul,1990) andreject allogeneic skingraftsfaster than males (Butterworthetal.,1967;Graffet aJ.,1969). Inaddition, it has been noted that
prevalenceof predispositiontoallergy changes ataboutageIS from malesto females (Scbuurs and verheul.I990).
The evidence thar thereis a differencebetweenthe immune system of malesand females suggeststhat theinfluence of gonadalsteroidsis very important. Muchof the workdone has been ondetermining the influence of these hormonesonvarious arms of theimm uneresponse.
It isimportanttonote herethat the outcome of in vivo studies donein various laboratories is influencedby the dose andtypeof administration of the hormone as well asbyage of theanimals used (Ahmed er01.,1985).
1.%.%HumoralImmuni ty
1.%..%.1 DirKt Acti on or Gonadal Horm ones
Estrogen hasbeen showntoenhancetheimmune response tovarious antigens in severalspecies(StemandDavidson,195 5~Kenny andGray,1971~Myersand Petersen, 1985;Erbachand Babr.,1988). Nikolaevichet at.,1991demonstratedthatinjection of estradiol intoovari ectomized micestimulated the immune response as indicatedby increased Ab response to sheeperythro cytes, while injection of progeste ronedidnot have such an effect. Invitro studies indicatethat estrogen has a directeffect on theimmun e systembyinfluencinglym phocytefunction(Erbach andBah r, 1991).Kennyet al.,1976.
incubated" invitro,E.coli primed murine spleen cellswithvarious concentrations of estrogen. They foundthatphysiological levelsof E2caused an increase in the number
of A" secreting cells in male micecomparedtountreated cells as demonstratedin a haemolytic plaqueassay(P FC ),byexamining the ratio of rota! plaquesfrom E2 treated cellswith controlcells.Theauthors suggestthat thepheno menon observedis independent of phagocyticfunction and that E2 directlyaffects lymphoidcellsin thespleen.
MyersandPetersen.,1985 ,found that intraperitoneal (i.p.)injectionofE2 in male ratsresultedinadose-relatedincrease in anti-sheep redbloo d cell (SRBC)antibody titres duringtheprimary immune response(characterizedbylbe productionofanri-SRBCIgM).
Unlike Kenny,these investigatorsfoundno increaseinthe number of Ab producingcells between contro land estradiol treated animalsas measured by a plaqueforming assay.
Similarly,inhumans,incubation of peripheral bloo d cells (PBL) with E2 caused an increase in the number of cells secreting[gM (paavonenetal.,1981) andIgG (Weetman etal.,1981).
Ainbenderetal.,1968,examineddifferencesInspecificAbto poliovirusinthe serum of males and femaleand found a differencebetween men and womenin the proportion of [gA and[gG polio antibody.Specifically ,itwas found thatin women the IgA polio antibody titre was equal toor greaterthan theIgG titre and thatin men it was less. Therewasno differencein theserumconcentration sof IgA or IgG betweenthe two groups.
KonstadouJakiset al.,1995,comparedinvitro andin vivoimmune parameters between female and malerats using a complicated experimental designinvol vin g honnone injectionsand gonadectomy.Theresults of the study consideringonlythein vivo [gG
10
levels in serum were asfollows:[10 levels in serum of male pupstreated with testosteron epropionate(TP)were increased as compared10controlsand[gGlevelsin serum of female pups treatedwith TP decreased;gonadectomyin bothsexesproduced effects similar to those as animals treated with TP.Thusinmale rats the increaseofIgG productionin vivoafter castration indicates that testosterone suppressesT and B cell functions.
Anotherstudyexamined thein vuroeffectof physiolo gical concentrations ofE2 andtestoste ron e(Te) 00 Agnon-specificdifferentiationof human peripheralblood mononuclearcells(pBMC) stimulated withpokeweed mitogen (PWM). Assessmentof B cell differentiation was indicated bythe number of Ab secreting ceJlsassayedby reversehemolytic PFC assay using SRBC as indicatorcells. IIwasfoundthatin vitro differentiation ofB cells.from both men and women.stim ulated with pokeweed mitogen and cultured withphysi ologicallevels ofE2wassignificantlyaugmentedas compared to controls and that physiological concentrations of Te inhi bi ted PWM·induc:edB cell differentiation(Sthoeger eraJ.•1988 ).
The variable effects of estrogen on the humeralim m une response isillustratedin the observationthat production of Ab toCandida albtcans in mice is enhanced by E2 at low levels but depressedat high levelsofthe hormone (Mathuret al.,1979).
Similarly ,itbas beenfound that women havebetterAb response10the Hepatitis Bvaccineth.anmendo asindicatedbythegreatergeo metric meantiters for anti·HBs (Struve<IaJ.•1992).
11
Evidence forthe direct effect of sex honnoneson the immune responseis also supportedbythe fact that receptors for estrogen have beenfoundinmouse spleen (DetlefsenetaJ.•1977).human. spleen(Danelet al.;1983;Stimson.1988) and onhuman PBL's(Daneletal.•1983;Weusten et al.•1986).
1.2.1.1 lDdirect Actio.of Go.adaJHormones
Severalpapers.illustratingboth in""'"andinVItroexperiments, have indicated that the influence of hormones on humoralimmunity is indirect (paavovnenet al.•1981;
Barneset 01.•1974;Stimson andHunter, 1976;Holdstocketal.;1982).
Erbach and Bahr, 1991.investigated the requirement ofthe thymus in the enhancement of humoral immune responses byestrogen. Adult femaleratswere ovariectomized andthymectomizedorsham. thymectomized and s.c.given E2 or left untreated.Results showed that E2enhancementofinvivohumoralimmunityrequires the thymus.Both male and femalerats need anintactthymus to achieve E2 enhancement of Ab titres.
1.2.3 Cellul arImmun ity
Estrogens and progestinsinhibitproliferativerespo nses of T cells to certain mitogens and antigens [phytohaemagluttin(PRA),Con A or purified protein derivatives (Mendelsohnetal.,19n;WyleandKent.1977)]. Athreya etaJ.•1993, evaluated the effectof thepresen ce or absence(controls)of varioussex steroids00theproliferative
12
responsestoPHA.n.~2oranti·C D3 ofPBMe s taken fromnormal human adultmales and foundthatE2 and Te bad no consistenteffect on the proliferative responsetoanyof thestimuli tested E2+PHA causedadecreaseInthepercentageofCD4·Tcells and E2+IL-2 an increaseinthe numberof
cos·
T cells.Te+1L·2 increased thepercentage ofCD4·cellsindicating a potentialrole of hormonesinTcell activation. Incontrastto this,Gilbody etal., 1992, studieddoserelated effects of estradiol00ratthymicand splenic T lymphocyte responsiveness to mitogeos andfound that estradiolstim ulated responsivenessof lymphocytes.Konstadoulakis et al.,1995. didinvitrostudies enrats and thecellular response during selfMLR(p.IO). Theyfound that in W\lO administrationof Te had anegative effecton self~andE2injectionhad apositive effect as shownbyan increase in
~after castrationand a decrease in~after oophorectomization.
Bara!etal.;1996,found that pretreatment of PSIS target cells (8 murine mastocytoma cell line) with esttadiolor tamoxifen (estrogen antagonistand potent immunomoduIa tory agent) makes them significantlymoresuscep tibleto Iymphokine activated(LAK) cellmediated cytotoxicity.
1.2.4 The Cycleand ImmuneResponse 1.2.4.1 HumoralaodMucosal Immunity
Studies done in miceandratsin dicate that the levelsofAb's fluctuateduring the estrous cycle. Total [gA levelsinratuterine secretions obtainedbyuterineflushing are
13
higherduring theestrusand proestrus when comparedto thediestrus;total IgG levelsIn uterine secretionsare higherintheproestrus phaseofthe cycle as comparedto theother phases(Winand Sandoe,1971,1980). Inaddition. migration oflymphocytesto the genitaltract.specifically the uterus. vagina andcervix,infemale miceis influencedby the estrous cycle;it has been foundthat thereis an increasednumberofIgAprod ucing plasma cells atthese sites during proestrusandestrus(McDermottetal.,1980;Rachman eta/.,1983).
Severalgroups haveshownthatpro gesterone andestrogen directly influence the fluctuating levels ofsecretory component (SC) and antibodies, as wellas thenumber of B cellsinthegenitaltractof female ratsand mice(Wang et al.,1996;wrreand Sandoe, 1977;Sullivan et al.• 19 &3). Similarly,cyclical changesinSC,measured inuterine secretions,and total Ab levelshave been demonstratedin humans (Sullivanelal.,1984;
UsalaetaI.,1989).SpecificalJy,Usalaet al.,1989,investigatedimm unoglobulinlevels In cervicovaginaJ secretions during the menstrual cycle using the microradial immunodiffusionmethod.Results indicated that IgG levelsinvaginalfluid samples were high inthe follicularphase witha steadydecl inetolow levels throughouttheluteal phase.
IgA waspresentat levelsIn-fold less thanIgG andneartheassayslimitof detection tho ughit was seen that the lowest vaginal concentrationwas aroundthe midlutealphase.
Severaldifferent systemicroutesof administration(s.e.,i.m.andi.p.) ofantigen havebeenshown toinduce specificAb'sinthe femalegenitaltractof severalspecies includinghumans (Gallichan and Rosenthal,1996;Miller et a/.• 1992;Ograand Ogra.
14
1973;Parr and Parr. 1990).
Intravaginal and intrauterine imm uni zation aremucosalro utes of immunizationand havebeenableto induce both specific(gG and 19Ainthe genitaltract,vaginalsecretions, andinserum(Milleiganand Bernstein. 1995;Ograand Ogra.,1973).Other mucosal sites, for example. intranasal(i.n..)generatesboth IgGand IgAin thevaginal secretions of the genitaltract(Gallichan and Rosenthal,1996;Musteret al.,1995).
Fewofthesestudiestake into account thestage ofthe c;;ycle.Gallichan and Rosenthal,1996.examined the influenceoftheestrous cycleinmiceon the levelsof Herpes SimplexVirus Type2 glycoprotein(HSV-gb ) specific IgA and IgG Ab',inthe femalegenitaltract.specificallyvaginalfluid. The mice wereimmunizedi.n. with AdgBl.arecombinant adenovirus type Svector. Resultsindicated that therewas a cyclicalfluctuationinthe Ab production,JgG was highestin diestrus andlowest atestrus, IgAwas highestinestrus and lowestindiestrus.Susceptibilityof uaimmunizedmiceto intravag inalHSV-2infection wasstudied duringvarious estrous cyclestagesandit was found that mice in fect ed duringestruswereprotected from infection since no viral replicationor genital pathology was observed, Inaddition.resultsshowed that i.n.
immunization duringaprogesterone-induced diestrus-likestate protects against subsequent intravaginal challenge assignificantly less virus replicationandgenital pathology was observed.Thediscovery that levelsof IgG and IgA antibodiesinthe genitaltractof i.n.
immunized micevary inverselywitheach other and are dependent on thestageof the cstro us cyclereflects the changesthatoccurinthereproductivetractduringthecourse of
IS
the cycle.Estrusisthe timeof mating and it follows thathighlevels ofIgA should be presenttotakecare of the pathogens associated with mating (Gallichan and Rosenthal, 1996).These increasedlevelsofIgA seenat estrusare supportedbythefindingthat there isanincrease in the number ofIgAprod uc io g plasma cellsintheuterus.vaginaand cervixduringproestrusand estrus (McDermott et01.,1980;Rachmaner aJ.,1983)andan increaseinSCin theuterus during estrus (Sullivaneral.,1983).
Simi larly,Parr et al.,1994investigated theeffectof the estrouscycle and sex ho rmoneson vaginalinfectionof adult mice by HSV-2.Results indicated thatinoculation dwin gestrusresultedinno infectio nwhereas inoc ulatio nintheprogest eronedominate d phase resulted in mice becoming infectedwiththe virus(panet al.;1994)
1..1.4..1 Prosta llaodial
Prostaglandins(pG)are also involvedintheimmuneresponse.Theyaremediators atseveral levels(Kunkel,1988). Leslieet al.• 1987notedthat female mice synthesized 8greateramount ofPGthanmal e mice and with greatervariationbetween femalesinthe group,therefor eleadin gto thehypothesis thatPGsynthesiswasmodifiedbyhormones durin gthe estrous cycle. Alater study found thathormo nal status of humanfem alesis associatedwithchangesinPGmetabolism. Leslie andDubey , 1994, foundthat both
~andPGI:wereincreasedinthemedi umof cultured monocytes isolatedduring the luteal phase compared to medium fromfollicular phase cultures and to monocytescultured from males donors.
16
1.2.4.3 CeUular Immunity
Herrera et al.;1992. examined the response of human lymphocytestomitogenic stimulation over the course of the menstrual cycle andin response toin vitroaddition of physiologicalconcentrations of E2 and P.Thestudyshowed that therewas large intra- individual variation over the course of the menstrual cycle and that invitroadministration of estradiol and progesteroneatphysiological concentrationsinhibited prohferation of male aod female PHA stimulated human lymphocytes.
Similarly,Bjune, 1976. investigated in vitro lymphocyte responses to PHA during the menstrual cycle and pregnancy. Inthisstudy,2 healthy women were bled twice weekly for 2 months.Response ofPBMetoPHAwere recordedISincorporation of~.
thymidineinthe presence of 10% autologous plasma and 10% pooled human serum. One pregnant woman andtwomales served as controls.The results indicated that in thetwo cycling women,PHAresponses varied greatly throughout the cycle.They found that at the time of ovulation the responses were low and that during mensestheywerehigh. They also foundthataddition of autologous serum resulted in strong suppressionin the pregnant woman andincycling women in theluteal phase of the cycle.whereasit had an augmenting effectinthe follicular phase. The PHA responsestoautologous plasma were constantinthe males and the pregnant woman.
Incontrast, anotherstudy investigated nine women over the course of one menstrual cycle measuring reactivity of blood lymphocytes to PHA during the menstrual (low levels of E2 and P),midfollicuJar (peak levelsof estradiol), and midluteal phases
17
(peak. levels of progesterone). Data indicated that there were no differences in reactivity to PHA over the three menstrual cycle phases and no relationship with any of the hormones measured (Caggiulaet aI.,1990).
Inaddition to studies done on the PHA response during the menstrual cycle, a recent study has been done on cytotoxic lymphocyte (Cl'L) activity during the menstrual cycle. In thisstudy
en.
activity was monitored in the reproductivetractin relation to the menstrual cycle. Human female reproductive tract cells were isolated from hysterectomy patients from the fallopian tube, uterine endometrium, endocervix, ectocervix and vaginal mucosa. The cells were cultured overnight in the presence of fi..-2 to yield effector cells and then these effector cells were incubated with or withoutOKD(anti- CD3 mAb) in a chromium release assay. This assay measured totalCDr T cell mediated lytic activitybyuse of FcRIIllll bearing,51Cr-Iabelled PSIS target cells. Results indicated thatcor
CDS"cytolytic T cells are found throughout the reproductive tract and,when the proliferative phase (follicular) and the secretory phase (luteal) were compared, that the capacity for CD3"T cell cytolytic activityinthe uterine endometrium is present during the proliferative phase but absent during the secretory phase. This indicated thatCOl'CDS" cytolytic T cells are hormonally regulated. Itwas also found that in postmenopausal women, the entire reproductive tract retains the capacity for strongcor
T cell cytolytic activity.The authors suggest that the data support the idea that high levels of E2 and P presentinthe luteal phase of the menstrual cycle downregulate CTL activity in the uterus thus allowing for implantation of the semiallogeneic embryo.They18
also conclude dlat CTL activity is regulateddifferently in different regions ofthe reproductivetract(WhiteetaI.•1991)
Fluctuationsin theWBC counts hevebeenobserveddurin g the menstrual cycle ofbumans.MathureraJ.,1979 investigatedcyclicvariationsinwhitecellsubpopulations inserialblood samplesfrom males and females. Monocytesand granulocyteswere increasedin theluteal pbase as compared to thefollicularphase;WBC.totalTcells and lympbocyt ecountsweredecreased at the pre-ovulatoryE2pealeinthefollicular phase.
Theconclusion of Mathur et al.,1979wasthatlympbocytecountswerenegati vely correlatedwithE2 andmonocyte andgranulocyte countswer e positivelycorrelatedwith progesterone. Results also showed that there were no cyclical variations inwac populationsin men andin womenwithnonovulatory cycles.
Therehas alsobeensome interestinwhether theimmune response differencesthat areseenbetweenmalesand females existatthelevel of extrathymic T cells ,definedas T cellsexpressingTcell receptor. (CD3) ofintermediate intensity {i.e.intermediate Teells) and a bigh levelof 1L-2receptor fJ-<;bain.invarious organsof mice. Itwasfowles that the C04·'C08 "T cells ratio is higher in females as comparedto males and that intermediate Tcells ofpossiblyextrathymicongin were more predominantin the liverand otherorgansof femalemicethaninmales (KimuraetaJ.,1994 ).
Sex hormones play a role inregul ating antigen presentation by uterine cells (obtainedbyuterienzymati cdigestio n)infemale ratsas demonstrat edbyWiraand Rossoll.Antigenpresentationwasmeasuredbyovalbumin-sensitized T cellsculturedin
19
the presence ofirradiated epithelialor stromal cellsand ovalbuminwith proliferation measuredby)Hthymidineuptake.Resultsindicatedthat antigenpresentationbyuterine epithelialcells ishigh atdiestrus.lowat estrus. Antigen presentationbyuterin estromal cellswashighatestruS,lowatdiestrus.Inaddition. if estradiolisgiven to ovariecromized rats this causesanincreaseinantigenpresentation bythe epithelial cellsand a decrease inantigen presentationbystromal cells ascomparedwithcontrols(Wira andRo sse ll, 19 95 ).
1.25 Con clusioas
Itis apparentfrom thevolume ofwork thai hasbeendoneon theinfluence of gonadal hormones on the immune system that thereismuch interestinthe area butthe pictureis still unclear. Contributing 10thisphenomenonisthe knowledgethatmany investigationsyield differentresults dependingon theroute of administrationanddose of the hormonesused forstudies.Aswell,different resultsare obtainedfrom differentsites including uterine.vaginal,mucosal,andsystemic areas of the system. Although the resultsdifferin thesecircumstancesit has been shown conclusivelythat gonadalsteroids do indeed influence the immuneresponse inhumansand animals.
20
1.3 Cytokines
Cytokines arepeptides or glycoproteinsthat act as soluble mediators of immune response. They regulate local and systemic immune and inflammatory responses as well as many other biological processes (Oppenheim et al.,1994).
They are highly potent molecules that act at concentrations of 10.10to 1O'1SM influencing the immune system as autocrine, paracrine and endocrine hormones (Oppenheim et al.,1994).
All the information in the following sections on cytokines has been obtained from two primary sources j.g, Abbaset al.,1994, and Oppenheimet al.,1994.Due to this fact the publications will not be sited throughout the text.
1.3.1 Interleukin 1
Interleukin 1 is a cytokine that is principally produced by activated mononuclear phagocyctes but can be produced by other cell types.These include:B lymphocytes, natural killer cells (NK),T cell clones grown in culture,keratinocytes, dendritic cells, astrocytes, fibroblasts, neutrophils,endothelial cells and smooth muscle cells. Significant amounts ofIL~I have been found in skin,amniotic fluid,sweat and urine.
Lipopolysaccharide (LPS),a bacterial cell wall product,tumour necrosis factor and interleukin 6,other cytokines,and CD4+T cells interacting with antigen presenting cells, can all initiate production of IL-I by mononuclear phagocytes.
The principle function of IL-l is as a mediator of the host inflammatory response.
21
Theactions ofIL· ldependon the concentration of IL-I presentin the system.
Atlow concentrations of IL-I, [1..1acts on mononuclearphagocyt esin an autacrinefash..ioatofurther stimulatesynthesis of IL·land to inducesynthesisofIL--6 It alsoactson mononuclear phagocyctesto synthesize chemckines, molecul esthat stimulate and direct leucocyte movement
When lL-1is presentat higherconcentrationsit can exert endocrine effectsby causing fever,synthesi s ofacute phase proteinsand cachexia.
fL·I exertsitsactions through two distinct forms, those of IL·laand IL·lJl Thesetwotypesofa-I arepeptidesthat areencodedbyseparategenesandshare26%
aminoacidsequence homology. Although theseare distinct forms of0..-1theyare virtually identicalinpotencyandactivity.
Inadditionto havingtwodistinct forms ofIL·Ipresentinthe system there isalso anaturallyoccurrin ginhibitorofll..-l. This inhibi to riscalledn.·l receptorantagonist (IL.Ira)and actstoinhib it IL·lbybindingto IL-I recepto rspreventingthe functional[L.
Ifrom bindingand exertingit'sinfl uence. Ils-Irais a competitiveinhibitorthatactsto endogenousl yregul ateIL-Iinthesystem.
lL-la and IL· lfJbindto high affinityreceptors (K.._l0-10M)that are found on all IL-Itargetcells. Two distinct types of receptors, IL·IRIand n.·IRll.have been found to bindtoa-Ia and IL-lfJ equally. Thesereceptorsare28%homologous inseq uence similarity and have three extr acell uardomains.
[1..1RIisthetypeI receptorandit has a217 a.a. cytoplasmictail that can transmit
22
signals to the cytoplasm of the cell when lL-I is bound.
lL-IRII is the second distinct type of receptor found that will bind lL-I and. unlike [l-IRl.it does not have a cytoplasmic tail. Thus it cannot transduce signals to the interior of the target cell.
Itis also important to note that one of the major functions of IL-I Rll is thought to be as an endogenous inhibitor of lL-11J at inflammatory sites duetothe release of the extracelluar domain of the receptor into solution at times of inflammation. This soluble lL-IRll can bind lL-IP more strongly than lL-la.
1.3.2 Tumour Necrosis Factor a.
Tumour necrosis factorais a cytokine that is primarily produced by activated mononuclear phagocytes but can also be producedbyantigen stimulated T lymphocytes, activated NK cells and activated mast cells.TNFais the mediator of response to gram negative bacteriawiththe response due directly to the presence of LPS in the bacterial cell wall. The effects ofTNFa on the systems of the body depend largely on the concentration of TNFa present.TNFa. can act in an autocrine,paracrine and endocrine manner and has effects in much the same way lL-I does.
At low concentrations TNFa acts locally as a paracrine and autocrine regulator of leukocytes and endothelial cells. TNFa.., at these low concentrations. causes activation of neutrophils;increase in expression of adhesion molecules contributing to accumulation of leukocytes at the area of inflammation; production of other cytokines like [l-I,IL-6 and
23
TNFaitself as well as chemckines;and augmentation of expression of Class I MHC molecules thereby assistingincytotoxiclymphocyte (CTL) mediated killing of infected cells.
At high concentrations.TNFaactsinan endocrine manner and enters the blood stream. WhenTNFais acting in this manner it produces different effects on biological systems. These effects include the induction of fever by acting on the hypothalamus.
induction of mononuclear phagocytes to secrete ll.-I and fi...6 into circulation.increased secretion of serum proteins like serum amyloid A protein thus contributing to the acute phase response,activation of the coagulation system, and suppression of bone marrow stem cell division.
As with IL·l ,two types of high affinityrecepto rs have been identified to bind TNFa. The type Il receptor has a higher affinity forTNFathan Type I. Each receptor is composed of a large extracelluar domain.a hydrophobic transmembrane region and an intracellular transducing piece. Itis thought that thetwotypes of receptors induce different effector functions. Binding ofTNFato Type I receptor promotes cytotoxic activity and Type Il promotes T cell proliferation.
1.3.3 Interleum 6
lnterleukin 6 is a cytokine that has a wide variety of biological effects. Itis mainly produced by mononuclear phagocytes,vascular endothelial cells and fibroblasts.
The secretion of IL·6 is often in response to ll.-I andTNFasecretion.
24
IT.-6 synergiz.es
n..(
andTNFa to induce the acute phase respo nse. ll.-6 participatesinthis response by inducing hepatoeyetestoproduce serum proteins like fibrinogen.Itis also knowntoserve as a growth factor for activatedBcells.Itenhances Bcell replication, differentiation., andimm unoglobulin production.IT.-6 has the abilltytosynergizc with ll.·1 and TNFa augmenting the mitogenic effects ofthese cytokines on T helper cells.This effect isinpart due to theabilityof[1..- 6to increase expression of1L-2receptors.
The high affinity receptors for IL-6,(K.t-IO·IO-10.1:M),are expressed on a variety of cell types.These include macrophages, myelomonocytic cell lines, hepatocytes,resting Tcells, activated orEBVinfectedBcells,and plasma cells.The receptor is composed of an a anda
JJ
chain.The ll.-6Rachain does not have a cytoplasmic domain. This chain binds[1..-6 withlow affinity producing theIL-6Ra-IL-6complex which then binds withhighaffinitytoIL-6Rllchain. This chain does contain a cytoplasmic tad and thus the signal can be transduced.1.3.4 lDterieukio 2
ll..-2is the principlecytokine responsible for the growth ofTcells,specifically the induction of T cells to move from the G)phase to the 5 phase of the cell cycle. This cytokineis producedbyT cells, specifically CD4-T cells but alsobyCDS-T cellswhen antigen and costimulatory factors are present.
[1..-2 has the ability to act in an autocrine and paracrine manner.Itfunctionsinan
2S
autocrinemannerbyacting as a growthfactorfo r theTcellsthatproduc eit but can also actonnearbyTcellstoinducen..·2thusactingin a paracrine manner. Itdoes not circulateinthe blood during animmune response thus does notactasan endocrine growthfactor.
Becauseofitseffects on Tcells,amajorcomponentoftheimmunesystem . itis thought to be a critical immuno regul atorycytokine.Itinfluenceshwnanlymphocytes to promote not onlyproliferationbut alsoproductionofotherlymphokines likeIFNy, TNFl\, IL-6, 1L-4.lL-l. IL-S,and GM-CSF. IL-2 will alsostlmulate NK cells toproduceother cytokinesthat intumactivatemacropbagesand enhancethecytolyticactivity ofNKcells.
Theeffect of fi.-2 on NKcells producea superior killingcell called theIymphokine activatedkiller cell(LAK).IL-2 influencesB cells as wellbyenhancingproliferationand antibodyproduction,preferentiall ypromotingproductionofIgG2antibodi es.
The IL-2 receptor is composed ofthree polypep tides,a,
P
and y. Each polypeptideon its own can bind IT.-2 butwith lowaffmity. Togethertheybind witha highaffinity<K..-IO·"M).Theachain hasonly a small cytoplasmicdomain and thuscanno t transduce signalstothe interiorof the cell. The
P
andychains havelarger cytoplasmic domains andcantransd ucesignals totheinteriorofthe cell.1.3.5 laterieukin 4
Interleukin4isa cytokine thatisprincipal lyproducedbyCD4+T lymphocytes,
26
specificallythose oftheT82subset. Itis alsoproducedbymast cells. lL-4 was initially describedas aBcellgrowthfactor asit helpsinBcellproliferationandas aBcell stimulatory factorbecause of its abilitytoinduce Class
n
MHC expressionon restingB cells. 1L-4is a major regulator of heavy chainclass switching as itisrequiredfor the production of IgE antibodies.Inaddition, 1L-4 influencesexpression oflow affinity Fe e receptorsonBcells.IL-4actson macroph ages toinhibit activation and block the effectsof another cytokiae,lFNy,produced bythe THI subset JL.4actsas agrowthand differentiation factorforT82cells andsuppresses the induction and fimctionofTHIcells.
IL-4 also influences expression of some of the adhesion molecules like VCAM·l on endothelial cells andca uses endothelialcellstosecreteMCP·I,a chemota cticprotein.
These functions allow forinflamm atory reactionsthal arerich in monocytes and eosinophi ls.Mastcells areinfluenced byil.-4asitpromotesgrowthofthistypeof cell.
il.-4,through aU thesefunctions, seems to be a principlemediato rof allergic reacti on s and, as itis characteristicallyproducedby theTH2subset. is responsible for the prevention of many cell-mediated immun e responses by preventin g theTHI subset functions.
1.3.6 laterferooy
lFNy isproducedbyboth naive(T.O) and T.lCD4-helperT cells.byCDS"T cellsandNK.cells when activatedItupregularesexpressionofClassI and ClassII MHC
27
thus aiding in cytotoxic killing and promotin g antigen presentation to CD4-T lymph ocytes.It acts asa potentactivator ofmononuclear phagoctyesinducingthe microbicidaland cytotoxicactivityof macrophages and production of othercytoki nessuch as lL·t, IL-6,IL-S,andTNFa. NK cells,neuuophils,andvascular endothelial cells are alsoactivated bythiscytokine.
IFNyactson T and Blymphocyt es to promotedifferentiation.specificall ylFNy promot es differenti ation of naiveTcells to the Tal subset and inhibitsproliferationofthe TR2 subset. CDS·cellsare aidedinmaturationbylFNyand Bcellsexposed to IFNy produceIgG2aand IgG)subclasses ofantibodies preventingproduction ofIgG IandliE.
The effects of IFNy described above all contribute to cell mediated immune responses and thesuppression of humoral mediated immune response.
1'" Su Hormonu a.ad Cytokincs
Thereareseveralstudiesthatindicatethe infl uence of sexhormoneson cytokine production.Thisinfluence has notbeenexp lained althoughagreatdeal ofevidenceexists for therelationsh ip.Muchof the work has been done on theinflammatorycytokine It·1 andsome information is available on several other cytok ines. Thefocus of this section will be on the cyto kines measuredin this study.
1.... 1 Ses Hormones aad
n-i
Oneof the earliest studiesdoneto show the influenceof gonadal steroi d on
28
cytokine productionwasbyCannonand Dinarello in1985.They examinedlL~1activity as measuredbythe effect onproliferati on of0I0 cells,amurineTcell line.stimulated with PHA.WhenJL.I activitywas assayedusingplasma from health y humanfemales.
there wassignificant Ila-l activity in theplasma isolated duringthelutealphase as comparedto the fo llic ul ar phaseof the menstrualcycle. Subsequently.anexperim ent donewith plasmafrom fivehealthywomenwhowerebled onceduringthe follicular and luteal phaseoftheir cycle(as determined by pro gesteron econcentrations)showed that the luteal phase plasma had greaterlL~1 activitythan thefollicularphaseplasma. They postuJatedthat this increas einIl>tactivityduring the luteal phase ofthe menstrual cycle wasconsistentwiththe0.2 to0.6°criseinbody tem peraturethatis seenintheluteal phase andtheinflamm atoryfocuspresentafter follicular rupture (Cannon and Dinarello, 1985 ).
Sincethen.therehavebeen severalstudiesdoneon the infl uenceofgonadal steroidson theproduction ofcytokin esboth bybumans and animalsinbothinvitro and invivosystems.
Huet al.•1988examined theeffec t of estradiol on Ils-lsynthesis/sec retion byrat peritoneal macrophagesas measured by a thymo cyteproliferation assay.Using peritoneal adherentcells (PAC)iso latedfrom fivefemaleand malerats they analyzed theamount of
n..t
spontaneously secretedinculture and found thatcellsfrom adult femal es secreted morelL~lthancellsfrom malesorprepubescentfemal es. Inaddition.ovariectom y led toredu ced synthesis of lL·IbyPAC andinvivoestradiol administeredsubcutaneo usly29
