An- - AG 4. PERFORMING ORGANIZATION REPORT NUMBERIISj S. MONITORING ORGANIZATION REPORT NUMBER(S) University of Washington NA Sa, NAME OF PERFORMING ORGANIZATION 6b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION jf iim0Kibit)1 University of Washington I AOffice of Naval Research &L ADRES (Ci Stte, na ZP Coe) b ADDRESS (City, State.. aria ZIP Code)- Harborview Medical Center 800 N. Quincy Street 325 Ninth Avenue Arlington, VA 22217-5000 Seattle WA 96104 _____________________ Sa. NAME OF FiUND#NGJSPONSORINYG 3b. OFFICE SyMBOL 9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER ... RGANIZATION i 4 oQpwiawe) Office of Naval Research ORN00014-87-K-0369 Sc- ADDRESS (City, State. aria ZIP Code) 10 SOURCE OF FUNDING NUMBERS ZROGRAM ?ROJECT I ASK NORK UNIT 80 unySre ELEMENT NO 1"0 NO !JOACCESSION NO. Arlington, VA 22217-5000 61153N I RRU4108 ,"TE(Includ Securir Clawsricat"on j 1 (U) Influence of Stress-induced Catecholamines on Macrophage Pacyoi 12. PERSONAL AjTHORS) 1 Itamar 6. Abrass Christine K. Ab rass 13a. TYPE OF REPORT i'3b, TIME COVERED 4 DATE OF REPORT (Year. Monlth. Day) 115 PAGE COUNT Annual FROM //8o2318 Api 1,99 1SUPPLEMENTARY NOTATION 17 .2SATI C_..DES 18 SUBJECT TERMS (Continue on reverse ir necessity aria odentety by 0oick fumor? 4 -LD OU .- fiROUP ________________________Stress, macrophage, phagocytosis, adrenergic 19 A8STR.ACT 'C,)nt,nue on reverse or necessary aria identity 0.v niOcknuorraro (Zr-. beta-adrenergic receptor concentration and e e las ctiv ty change with mrophage activation. Resident (R) and thiogly ollate (TG)- r imulatemarhge (M0) have comparable concentrations of beta-adr nergic rece or 213 6 - 143 and 2110 4orf-YAK327 sites/cell respectively), while Bacillus Calmette G in (BC ;activated macrophages have 62% fewer beta-adrenergic re tors (808 _190 sie te W~ . The increase in adenylate cyclase activity in TG- as compare to residentl& results in increased sensitivity to catecholamines. FcR-mediated phagocytosis is dependent on FcR concentration, internalization and concentration of FcR which in turn increases the availability of immune complexes for phagocytosis by the cell. Catecholamines also decrease internalization ado., 20. DiSTRiBUTOCN/AVAILAaLIY ::z ABSTRACT -" ABSTRACT SECURiTY C'-ASSFCATION I LNCLASSIFIED/tJNLIMircED:2 SAME AS RPT - :)7C USERS 22a NAME OF RESPONSIBLE i%:.i~fIDUAL :2b TELEPH4ONE (InClude Area C~ode) 9 22,. OFICE SYMBOL Dr. J.. Mad (2021694-405!,ON DODForm 1473. J UN 86 -'evious easons are osolet. :UFIjPTYCLASSiFiCATION OF THIS PAGE Si OlO*2-1-F-014-66O3
25
Embed
BETA-ADRENERGIC RECEPTORS DURING … · of beta-adrenergic receptors with both having greater than twice the number for BCG cells. ... in Table III. Both NaF- and isoproterenol-stimulated
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
An- - AG
4. PERFORMING ORGANIZATION REPORT NUMBERIISj S. MONITORING ORGANIZATION REPORT NUMBER(S)
University of Washington NA
Sa, NAME OF PERFORMING ORGANIZATION 6b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATIONjf iim0Kibit)1
University of Washington I AOffice of Naval Research
&L ADRES (Ci Stte, na ZP Coe) b ADDRESS (City, State.. aria ZIP Code)-Harborview Medical Center 800 N. Quincy Street325 Ninth Avenue Arlington, VA 22217-5000Seattle WA 96104 _____________________
Sa. NAME OF FiUND#NGJSPONSORINYG 3b. OFFICE SyMBOL 9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER...RGANIZATION i 4 oQpwiawe)
Office of Naval Research ORN00014-87-K-0369Sc- ADDRESS (City, State. aria ZIP Code) 10 SOURCE OF FUNDING NUMBERS
ZROGRAM ?ROJECT I ASK NORK UNIT80 unySre ELEMENT NO 1"0 NO !JOACCESSION NO.
Arlington, VA 22217-5000 61153N I RRU4108,"TE(Includ Securir Clawsricat"on
j 1(U) Influence of Stress-induced Catecholamines on Macrophage Pacyoi12. PERSONAL AjTHORS) 1
Itamar 6. Abrass Christine K. Ab rass13a. TYPE OF REPORT i'3b, TIME COVERED 4 DATE OF REPORT (Year. Monlth. Day) 115 PAGE COUNT
Annual FROM //8o2318 Api 1,99
1SUPPLEMENTARY NOTATION
17 .2SATI C_..DES 18 SUBJECT TERMS (Continue on reverse ir necessity aria odentety by 0oick fumor?4 -LD OU .- fiROUP
19 A8STR.ACT 'C,)nt,nue on reverse or necessary aria identity 0.v niOcknuorraro (Zr-.beta-adrenergic receptor concentration and e e las ctiv ty change withmrophage activation. Resident (R) and thiogly ollate (TG)- r imulatemarhge
(M0) have comparable concentrations of beta-adr nergic rece or 213 6 - 143 and 21104orf-YAK327 sites/cell respectively), while Bacillus Calmette G in (BC ;activated
macrophages have 62% fewer beta-adrenergic re tors (808 _190 sie te W~ . Theincrease in adenylate cyclase activity in TG- as compare to residentl& results inincreased sensitivity to catecholamines.
FcR-mediated phagocytosis is dependent on FcR concentration, internalization andconcentration of FcR which in turn increases the availability of immune complexes forphagocytosis by the cell. Catecholamines also decrease internalization ado.,
20. DiSTRiBUTOCN/AVAILAaLIY ::z ABSTRACT -" ABSTRACT SECURiTY C'-ASSFCATIONI LNCLASSIFIED/tJNLIMircED:2 SAME AS RPT - :)7C USERS
22a NAME OF RESPONSIBLE i%:.i~fIDUAL :2b TELEPH4ONE (InClude Area C~ode) 9 22,. OFICE SYMBOLDr. J.. Mad (2021694-405!,ON
DODForm 1473. J UN 86 -'evious easons are osolet. :UFIjPTYCLASSiFiCATION OF THIS PAGE
Si OlO*2-1-F-014-66O3
Security Classificatl-onof-this Page
digestion-of- FcR-bound imuwne complexes. Thus, the net- effect of catecholamines ontotal phagocytosis is theeconsequence of the balance of independent effects of-catecholamines on FcRconcentraton-and the efficiency of internalization anddegradation-of boundumateral. TG-10 as described above, are-the-most sensitive-to-the effects of catecholamines dch is confirmed functionally. TG-M0 demonstrate-the-greatest increase in FcR concentr to~n and inhibition of internalization-and digestion;resulting in no-net change,-in phagocy *s. In R-MO.and.BCG-M0 the effects oninternalization and digestion override themodest increase in FcR concentration; thuscausing a significant decrease in total phagocyi1-o-.-- C f4Lt
These observations show-that the influence of catecholamines on MO phagocyticfunction are complex and depend on the state of macrophage activation, regulation ofbeta-adrenergic receptor-concentration and adenylate cyclase activity, Fc receptorconcentration, and activity of post-FcR functions including rates of internalizationand digestion of FcR-bound material. Additional studies are needed to furtherunderstand the mechanisms involved in catecholamine-mediated regulation of macrophage-phagocytosi s.
T.copY
jCcession For
' L- U2Y Codes
S YiT
SECURITY CLASSIFICATION THIS PAGE
BETA-ADRENERGIC RECEPTORS DURING MACROPHAGE ACTIVATION
REGULATION OF BETA-ADRENERGIC RECEPTOR EXPRESSION AND ADENYLATE
CYCLASE ACTIVITY DURING MACROPHAGE ACTIVATION'
Itamar B. Abrass-0 and Christine K. Abrass-
*Division of Gerontology and Geriatric Medicine
Department of Medicine
University of Washington, School of Medicine
Harborview Medical Center
and
-Division of Nephroloqy and Department of Medicine
University of Washington, School of Medicine
Veterans Administration Medical Center
Seattle, Washington
2
ABSTRACT
Beta-adrenergic receptor concentration and adenylate
cyclase activity were determined on resident (R-MO),
thioqlycollate-stimulated (TG-M4) and Bacillus Calmette Guerin-
activated (BCG-MO) rat peritoneal macrophages. Beta-adrenergic
receptor concentration as determined by La---iodocyanopindolol
binding was: R-MO 2136±143, TG-MO 2110±327, and for BCG-M
808±190 sites/cell (mean ± SEM; p<O.01, ANOVA BCG-MO< R-MO and
Iso = isoproterenol. Values for basal cAMP and in the presence of
GTP were subtracted from values obtained for the other conditions
to determine the specific adenUlate cyclase activity for NaF and
isoproterenol stimulation. The results are expressed as the
group mean ± I SD of results obtained from indivicdtal experiments
(n).
* p < 0.01, analysis of variance, TG-M > R-MO and BCG-MO.
+ p < 0.05, analysis of variance, TG-MO > R-MO and BCG-Md.
20
I. INTRODUCTION
Fc-receptor-mediated phagocytosis of soluble immune complexes and antibody-coatedbacteria are important functions of macrophages. Phagocytosis of antigens bymacrophages plays a role in antigen clearance, as well as antigen processing forpresentation to lymphocytes which initiates cell-mediated and humoral immune responses.The purpose of this project is to study catecholamine-mediated alterations inmacrophage phagocytic function.
II. PROGRESS REPORT
1. Reculation of beta-adrenergic receptor expression and adenylatecvclase activity during macrophage activation.
Beta-adrenergic receptor concentration and adenylate cyclase activity were determinedon resident (R-M0), thioglycollate-stimulated (TG-M0) and Bacillus Calmette Guerin-activated (BCG-MO2 Eat peritoneal macrophages. Beta-adrenergic receptor concentrationas determined by- . I-iodocyanopindolol binding was: R-M0, 2136 ± 143; TG-M0, 2110 +327, and for BCG-M0, 808 + 190 sites/cell (mean ± SEM; p<0.01, ANOVA BCG-MO< R-M0 andTG-MO). NaF-stimulated adenylate cyclase activity wa§: R-M0, 35.4 + 6.6; TG-M0, 72.7± 13.7, and for BCG-MO, 32.4 + 11.9 picomoles cAMP/lO cells/min (mean ± SD; p <0.01ANOVA, TG-M0>R-M0 and BCG-MO), while isoproterenol-stimulated activity was: 16.0 +1.8, 31.2 + 3.8, 15.0 + 1.3, respectively (mean + SD, p<0.05, ANOVA TG-MO>R-M0 andBCG-M0). These data suggest that beta-adrenergic receptors and adenylate cyclaseactivity are differentially and separately regulated during macrophage activation.Adenylate cyclase activity is rate limiting, since resident and BCG cells reflect equalisoproterenol-stimulated activity in the face of different receptor concentrations.The data also demonstrate that TG cells are the most responsive to beta-adrenergicstimulation.
The manuscript describing these studies is attached.
2. Catecholamine-mediated regulation of Fc-receptor concentrationand Dhagoctic activity of rat peritoneal macrophages.
The influence of catecholamines on Fc receptor (FcR) concentration was measured atvarious stages of macrophage activation. Resident TG-stimulated and BCG-activatedperitoneal M0 were harvested and incubated with radiolabeled heat aggregated rat IgG(ARG). FcR concentration was determined by Scatchard analysis of binding data. As themodel immune complexes contain multiple Fc regions per aggregate and thus canparticipate in multi-site attachment, these measures of FcR concentration are onlymeaningful as a relative, but not an absolute measure of the changes in FcRconcentration that are associated with metaproterenol treatment.
EFFECT OF METAPROTERENOL ON NO FcR DENSITY
Metaproterenol (M)
0 1O-8 10- 6 1O-4
Macrophages (n)
Resident 3 24.9 28.7 35.4 42.9
TG 3 43.1 54.9 69.3 97.7
BCG 3 12.5 15.1 16.8 18.1
FcR density is expressed as sites (x 103)/cell. These results demonstrate that FcRdensity on TG-MO>Resident-MO>BCG-MO. In each case, there is a dose-dependent increasein FcR density with the addition of metaproterenol. BCG-MO appear to be lessresponsive to metaproterenol than resident or TG-MO, with TG-MO being the mostresponsive. Changes in FcR density were not altered by the addition of cycloheximidc(2 ug/ml), suggesting that the effect of metaproterenol does not require new proteinsynthesis. Prostaglandin E2 is released from MO following phagocytosis of immunecomplexes and like metaproterenol it activates adenylate cyclase. Binding studies wereperformed at 40 C after pre-treatment with metaproterenol at 370 C, thus it is unlikelythat internalization of bound material led to release of prostaglandins. However, toeliminate a possible role of prostglandin-mediated increase in cyclic AMP, cells werepre-treated with indomethacin (lO M). Indometacin pre-treatment did not alter theresults observed with metaproterenol treatment. These studies indicate thatcatecholamine treatment of Mo is associated with a dose-dependent increase in FcRconcentration.
The influence of catecholamines on FcR-mediated phagocytosis of model immune com lexeswas studied by incubation of R-MO, TG-MO and BCG-MO with radiolabeled ARG at 37U C andmeasurement of binding, internalization and digestion of ARG. In resident (21 vs 13%)and BCG (19 vs 11%) MO, lO- M metaproterenol induces a decrease in the total amountof ARG digested over 2 hours of incubation. In contrast to these observations,metaproterenol treatment of TG-MO is associated with no change in net digestion of ARG(45 vs 43%). As metaproterenol treatment induces a doubling of the amount of ARG boundto TG-M0 (as shown in the Table above), and a decrease in the fraction of bound ARGthat are internalized, there is no net change in the amount digested. In resident andBCG-MO, the effect of metaproterenol to decrease the rate of internalization/digestionof FcR-bound material is greater than its effect on binding; thus, the net effect is adecrease in digestion. These observations demonstrate that catecholamines effect morethan one step of FcR-mediated phagocytosis. Additional studies are needed to furtherdefine the influence of catecholamines on each step of phagocytosis and the majorsubclasses of FcR for IgG.
Eb*
A
3. AlDha-adrenergic receptor expression on rat peritonealmacroohages.
Studies described above demonstrated that rat peritoneal macrophages express beta-adrenergic receptors. However, endogenous catecholamines function as both alpha- andbeta-adrenergic agonists. Thus, it seemed possible that should macrophages expressboth alpha 2 - and beta-adrenergic receptors, catecholamine-mediated activation of bothreceptors might produce complex and even opposite effects. Resident, TG-stimulated andBCG-activated rat peritoneal macrophages were harvested and assayed for alpha-adrenergic receptors using [3H]-yohimbine. The absence of specific binding of [3H]-yohimbine to rat macrophages suggests that they do not express alpha 2 -adrenergicreceptors.
4. Beta-adreneraic receptor expression on mouse macrophaQe cell-lines.
Previous studies have evaluated net FcR expression and net phagocytic function of ratperitoneal macrophages. Yet, three major subclasses of FcR for IgG have beencharacterized on human, mouse and rat macrophages. These FcR differ in their aminoacid sequence, ligand binding affinity, trypsin-sensitivity, activation of secondmessengers, as well as activation of phagocytosis, enzyme release and other effects ofFcR-ligation. These characteristics of FcR have been most thoroughly evaluated usingmouse macrophage cell-lines. Monoclonal antibodies specific for two of the subclassesof mouse FcR for IgG are available. Thus, utilization of these reagents provides theopportunity to determine any FcR-subclass specific catecholamine-mediated effects onFcR function.
Mouse macrophage-like cell lines (P388D and J744a) were grown in culture and assayedfor beta-adrenergic receptor expression using standard methods.
P388DI £Ils do not express beta-adrenergic receptors; however, J744a cells bind 98fmole [ I]-iodocyanopindolol/per mg membrane protein. A macrophage (P388DI) thatexpresses FcR and is phagocytically active but lacks beta-adrenergic receptors thatwill provide a useful comparison for the J744a line which expresses both activities.Hybridomas for monoclonal antibodies to FcR II and FcR III (2.4G2 and 3G8 respectively,kindly provided by Dr. Jay Unkeless) are being cultured. The monoclonal antibodieswill be isolated and used to study catecholamine-mediated effects on each subclass ofFcR. Aggregated mouse IgG will be used as described above to assay phagocyticactivity. Thesp studies are in progress.