Peripheral Blood Lymphocyte Response to Acute Infections

Transcription

1 INCTION AND IMMUNITY, Apr. 1977, p Copyright X 1977 Americn Society for Microbiology Vol. 16, No. 1 Printed in U.S.A. Peripherl Blood Lymphocyte Response to Acute Infections in Humns JEFFREY D. THORLEY,* JAMES W. SMITH, JAMES P. LUBY, AND JAY P. SANFORD Deprtment of Internl Medicine, University of Texs Southwestern Medicl School, Dlls, Texs 75235; Dlls Veterns Administrtion Hospitl, Dlls, Texs 75216; nd the Deprtment of Internl Medicine, University of Texs Medicl Brnch, Glveston, Texs 77550* Received for publiction 30 September 1976 The response of subpopultions of peripherl venous blood lymphocytes to systemic bcteril or virl infections ws studied. B-lymphocytes were defined by the presence of surfce binding sites for mu chins, which were determined by immunofluorescent stining. T-lymphocytes were defined by the bility to form ctive sheep cell rosettes. Virus-infectible lymphocytes, which my represent ctivted T-lymphocytes, were defined by the bility to support virus repliction. Ptients with bcteril infections hd n increse in the B-lymphocytes of peripherl venous blood, wheres ptients with virl infections hd n increse in T-lymphocytes s compred to controls. The number of virus-infectible lymphocytes ws incresed in ptients with bcteril infections but not in ptients with virl infections. These studies suggest tht subpopultions of humn peripherl blood lymphocytes vry in response to different types of infectious gents. Humn lymphocytes cn be brodly seprted into B- or T-cells (22). Ptients with selective loss of B-lymphocytes suffer from recurrent extrcellulr bcteril infections, wheres individuls with selective loss of T- lymphocytes my suffer from recurrent virl infections (8). In ddition, ptients with virl infections commonly show n cute, trnsient diminution in cellulr immune responses, which suggests impired T-lymphocyte function (15). These observtions indicte tht the response of the humn lymphoid system to infection my vry with the type of infectious chllenge. However, one report demonstrted no chnge in the number of circulting B- or T- lymphocytes in ptients with bcteril nd virl infections (1), wheres nother reported tht ptients with infections hd ltertions in peripherl blood lymphocyte subpopultions (14). The present studies were undertken with vriety of systemic infections to clrify the peripherl blood lymphocyte response to infection in humns. This ws ccomplished by enumerting B-, T-, nd virus-infectible lymphocytes during bcteril nd virl infections. MATERIALS AND METHODS Subjects. Ptients hving infections cused by vriety of bcteril or virl gents were studied. During the time of study they were hospitlized t Prklnd Memoril Hospitl or the Veterns Administrtion Hospitl in Dlls, Tex. The durtion of infection prior to study ws estimted by historicl informtion obtined from medicl records. Ptients with bcteril infections (Tble 1) were studied for medin of 10 dys (rnge, 2 to 45 dys) fter the onset of illness. Their infections were predominntly cused by grm-positive pyogenic bcteri. Etiologicl orgnisms were identified by culture; the only exceptions were ptients with typhoid nd prtyphoid fever in whom etiology ws serologiclly proven nd ptients with lung nd brin bscesses in whom pproprite cultures were not performed. All ptients with bcteril infections were being treted with ntibcteril gents t the time of study. The men ptient ge ws 35 yers (rnge, 13 to 79 yers). Ptients with virl infections (Tble 2) were studied medin of 8 dys (rnge, 2 to 24 dys) fter the onset of illness. The virl gent ws isolted from 4 ptients nd serologiclly identified from 12. The other 12 hd typicl virl syndrome (e.g., heptitis, mononucleosis), nd no bcteril gent ws isolted from repeted bcteril culture ttempts. No ptient ws receiving ntivirl chemotherpy during the time of study. The men ptient ge ws 29 yers (rnge, 10 to 59 yers). Control subjects included 19 lbortory nd hospitl personnel nd 21 ptients who hd been hospitlized with high-risk pregnncies s tht resulted from hypertension. There were no differences in the numbers of peripherl blood cells between the personnel nd pregnnt hypertensive controls. The men ge of control subjects ws 29 yers (rnge, 16 to 43 yers). The rce nd sex distributions of controls were similr to those for ptients. Blood from controls ws run in prllel with blood from p- 110

2 VOL. 16, 1977 TABLE 1. Ptients with bcteril infections tht were studied Surfce Sheep Virus in- Dignosis/etiology immuno- cell ro- fectibilglobulin sette ity Endocrditis Stphylococcus Enterococcus Streptococcus Septic rthritis Gonococcus Streptococcus Pneumococcus Streptococcus Abscess Lung Brin Buttock Meningitis Meningococcus Pneumococcus Slmonellosis Typhoid fever Prtyphoid fever Gstroenteritis Pericrditis, Pneumococ cus Osteomyelitis, Stphylo coccus Peritonitis, Escherichi coli Fistul infection, Stphy lococcus Totl TABLE 2. Ptients with virl infections tht were studied Surfce Sheep Virus in- Dignosis/etiology immuno- cell ro- fectibilglobulin sette ity Virl Vricell-zoster Heptitis HB Ag neg HB Ag pos Infectious mononucleosis Rubell Encelphlitis, vricell zoster Vccintion, vccini Pericrditis, influenz Aseptic meningitis Disseminted cutneous vricell-zoster Herpes lbilis Virl myocrditis Totl BLOOD LYMPHOCYTE RESPONSE TO INFECTIONS 111 tients. Multiple studies were performed on some ptients nd controls. Blood collection, counting, nd seprtion. A 14- ml quntity of venous blood ws collected in ethylenediminetetrcetic cid. Peripherl blood leukocyte counts were performed in Neubuer chmber, nd differentil counts were done on 100 cells. The number of ech cell type ws determined by multiplying the totl leukocyte counts by the differentil proportion of ech cell type. One prt of blood ws mixed with three prts of sterile isotonic sline. This mixture ws lyered over 24:10 rtio of 9% Ficoll-33.9% Hypque nd centrifuged for 30 min t 600 x g (4). The cell suspension obtined ws wshed twice with minimum essentil medium (MEM) nd used for ll ssys. Morphologiclly it contined 85 to 95% lymphocytes. B-lymphocyte ssy. The suspension of lymphocytes ws incubted t 37 C for 30 min with 50 ml of 1:4 dilution of fluoresceinted got ntiserum in phosphte-buffered sline (ph 7.2), which is specific for humn mu chins (Meloy Lbortories, Springfield, V.), nd ultrcentrifuged prior to use. The ntiserum ws monospecific by gel rdil immunodiffusion t protein concentrtion of 10 mg/ml nd contined 4 x 106 Ag of fluorescein per mg ofprotein. After incubtion with the ntiserum, cell suspensions were wshed twice with phosphte-buffered sline, clered of erythrocytes by dding 9.85% NH4CI-Tris buffer, ph 7.65, nd then mounted on glss slides using nil polish-seled cover slips. Two hundred cells were scored under code s positive or negtive for membrne immunofluorescence. B- cells were quntitted by multipliction of the totl lymphocyte count by the percentge of immunofluorescence-positive cells. In some preprtions, ltex prticles were dded prior to incubtion to detect phgocytic cells tht were consistently less thn 15%. A negtive control ws performed with fluorescented norml got serum. B-lymphocyte ssy with incubtion. Lymphocytes were collected nd seprted s bove from five ptients with three types of cute infections: pneumonococcl pneumoni, stphylococcl endocrditis (2), nd heptitis B ntigen heptitis (2). B-lymphocytes were quntitted both before nd fter incubtion in MEM t 37 C in 5% CO2 for 18 h. T-lymphocyte ssy. T-lymphocytes were quntitted in smller number of ptients using the ctive sheep cell rosette ssy. This ssy ws chosen since it my more ccurtely reflect T-lymphocyte function thn the totl sheep cell rosette ssy (22). The ctive ssy does not mesure ll T-lymphocytes but mesures subpopultion. One million lymphocytes in 1 ml of MEM were incubted t 37 C for 15 min with 0.1 ml of 2.5% suspension of sheep erythrocytes (SRBC) in phosphte-buffered sline. The SRBC-lymphocyte mixture ws centrifuged t 1,500 x g for 10 min nd then incubted t 4 C for 1 h. The button ws gently resuspended, nd the cell suspension ws plced on glss slide with cover slip. Two hundred lymphocytes were scored under code s positive (three or more SRBC ttched to the lymphocyte membrne) or negtive (less thn three

3 112 THORLEY ET AL. SRBC ttched). No ttempt ws mde to quntitte "null" cells. Virus-infectible lymphocyte ssy. The number of lymphocytes tht could be infected with vesiculr stomtitis virus ws determined by incubting 2 x 106 cells in 1 ml of MEM with 108 plque-forming units of virus (Indin strin) t 37 C for 1 h to llow virus to penetrte cells (3). The cells were collected by centrifugtion, wshed twice with MEM contining 2% fetl clf serum (MEM-FCS), nd then incubted with 0.2 ml of rbbit nti-vesiculr stomtitis virus serum hving neutrlizing ntibody titer of 1:128 t 370C for 30 min to neutrlize extrcellulr virus. After three dditionl wshes with MEM- FCS, the button ws resuspended in 1 ml of MEM- FCS, nd 10-fold dilutions of cells in MEM-FCS were mde. A 0.5-ml portion of ech dilution contining 106, 105, 104, or 103 virus-infected lymphocytes ws mixed with n equl volume of wrmed 1.25% grose in MEM-FCS (nutrient gr) nd then lyered over monolyer of Vero cells. The finl wsh fter incubtion with neutrlizing nti-vesiculr stomtitis virus serum ws processed in the sme fshion to serve s control for the bsence of vible extrcellulr virus. After the gr hd solidified, the pltes were overlid with 3 ml of nutrient gr nd then incubted t 37 C for 48 h in 5% CO2 tmosphere. At the end of incubtion, monolyers were stined with 1.5 ml of 1:10,000 neutrl red in nutrient gr, nd monolyer plques for ech dilution of lymphocytes were counted. The results were expressed s the number of virus-infectible cells per 100,000 lymphocytes. Sttistics. The significnce of the differences of mens ws estimted by the nonpired t-test. RESULTS Blood counts. Peripherl blood leukocyte counts tken from 34 ptients during bcteril infections, 28 ptients during virl infections, nd 40 controls re summrized in Tble 3. Both groups of ptients hd greter number of circulting leukocytes compred to tht for control subjects. The incresed number of leukocytes reflected neutrophili in ptients with bcteril infections but reflected lym- TABLE 3. Peripherl blood leukocyte counts Peripherl blood leukocyte counts (men/ Peripherl mm3 ± SE) from: blood cell Bcteril in- Virl infec- Control fection tion Leukocytes 6, ,280 ± 520b 8,840 ± 960c Neutrophils 3, , b 4,880 ± 510 Lymphocytes 2, ,390 ± 160 3, c Non-B-cells 2,080 ± 110 2, ,130 ± 360b B-cells d 270 ± 50 Sixty-four determintions were mde from the control group, 55 from the bcteril infection group, nd 30 from the virl infection group. b Different from control t <0.001 level. c Different from control t <0.01 level. d Different from control t <0.02 level. INFECT. IMMUN. phocytosis in ptients with virl infections. There ws no difference in the number of peripherl blood monocytes, eosinophils, or bsophils between the three groups. B- nd non-b-lymphocytes. Ptients with bcteril infections hd greter proportion (men, 14 versus 9%) nd number of B-cells compred to control subjects (Tble 3). Ptients with virl infections did not hve different proportion (men, 8 versus 9%) or number of B- cells thn did controls. The number of non-blymphocytes ws similr in ptients with bcteril infections nd controls, but ptients with virl infections hd greter number of non-bcells thn did the other groups, ccounting for the incresed totl lymphocyte counts seen in these ptients. B-lymphocytes with incubtion. There ws no difference in the number of B-cells from five ptients mesured before nd fter n 18-h incubtion, indicting tht fluorescein-positive cells were indeed B-lymphocytes (Tble 4). T-lymphocytes. The percentges of peripherl blood rosette-forming cells in ptients with bcteril infections nd controls were similr to tht for the controls (24%), but ptients with virl infections hd significntly greter proportion of circulting, ctive rosette-forming cells (33%) compred to tht for control subjects (Tble 5). Virus-infectible lymphocytes. Ptients with bcteril infections hd greter numbers of circulting, virus-infectible lymphocytes thn did TABLE 4. Effect of incubtion upon proportion of B- lymphocytes B-lymphocytes (%) Dignosis/etiology Before incu- After incubtion btion Endocrditis Stphylococcus 9 9 Stphylococcus 8 12 Pneumococcus Heptitis HB Ag pos HB Ag pos 6 8 TABLE 5. Proportion of peripherl blood lymphocytes forming ctive sheep cell rosettes Group No. of determi- ntions Rosette-forming cells ntions (% SE) Control Bcteril infection Virl infection Different from control t the <0.01 level.

4 VOL. 16, 1977 control subjects or ptients with virl infections (Tble 6). The number of virus-infectible cells represented much smller lymphocyte subpopultion (20 cells per 100,000 in controls) thn ws mesured by the ctive sheep cell rosette test for T-lymphocytes (24,000 cells per 100,000 in controls). DISCUSSION Our studies confirm time-honored observtions tht the peripherl blood neutrophil count is significntly elevted during bcteril infections (10, 19) but is less so during virl infections (6). In contrst, lymphocytosis does not regulrly ccompny bcteril infections but hs been extensively described during virl infections, especilly infectious mononucleosis (18). Trnsiently reduced lymphocyte counts hve been demonstrted within the first week of experimentl infectious heptitis, but they re followed by lymphocytosis tht is more chrcteristic of virl infections (9). The nlysis of peripherl blood lymphocyte subpopultions is n pproch to understnding the immune response to infectious gents. Ptients with bcteril infections hd n incresed number of circulting B-lymphocytes, wheres ptients with virl infections did not. This grees with observtions by Niklsson nd Willims (14) but not with those of Aiuti et l. (1). Others hve demonstrted n increse in peripherl blood B-lymphocytes during virl infections (5, 7, 16). Most uthors reporting B- cells during infections hve not rigorously excluded cytophilic ntibody which my result in n overestimtion of the number of B-lymphocytes (11, 13). To void this, we quntitted only lymphocytes hving surfce mu chins, which ppers to more specificlly mrk B-cells (13). In ddition, overnight incubtion of lymphocytes resulted in no diminution of mupositive cells, suggesting tht these fluoresceinpositive lymphocytes did not hve surfce cytophilic ntibody. An overestimtion of B-lymphocytes due to cytophilic ntibody hd been TABLE 6. Number ofperipherl blood lymphocytes which were infectible with virus Virus-infectible No. of determi- cells Group no.ote (geometric men/105 lymphocytes + SE) Control Bcteril infection ± 2 Virl infection ± 1 Different from control t the <0.01 level. BLOOD LYMPHOCYTE RESPONSE TO INFECTIONS 113 demonstrted, prticulrly with gmm-positive cells (20, 21). Ptients with bcteril infections hd norml percentge of peripherl blood T-lymphocytes, wheres ptients with virl infections hd n incresed percentge. This increse in T-cells in ptients with virl infections ws reflected in n elevtion of the peripherl blood lymphocyte count. Others hve documented elevted T-lymphocytes during specific virl infections (5, 7, 14, 17). However, the proportion of peripherl blood lymphocytes estimted to be T-cells utilizing rosettes from nonimmune SRBCs hs vried from 35 to 80% in controls (2). We elected to use the "ctive" rosette test rther thn "totl" rosette tests becuse of the suggestion tht the ctive test is better reflection of T-cell competence (22). Lower vlues obtined with this test, however, mke ny inferences bout null cells impossible. Ptients with bcteril infections hd greter number of virus-infectible peripherl blood cells thn controls or ptients with virl infections. These cells my represent "ctivted" T-lymphocytes (3, 12). The proportion of peripherl blood virus-infectible cells ws so smll tht the increse in ctivted T-cells in ptients with bcteril infections occurred in the fce of norml totl numbers of T-lymphocytes. Conversely, ptients with virl infections hd norml number of virus-infectible cells in the fce of elevted totl numbers of T- lymphocytes. Thus, these studies indicte fundmentl difference in the response of the humn lymphoid system with different types of infectious gents. These findings re consistent with clinicl observtions tht relte infectious diseses to B- nd T-lymphocyte defects. ACKNOWLEDGMENTS J.D.T. ws the recipient of Public Helth Service trining grnt TOL Al from the Ntionl Institute of Allergy nd Infectious Diseses. LITERATURE CITED 1. Aiuti, F., M. V. Cirl, C. D'Asero, R. D'Amelio, nd J. Groflo Surfce mrkers on lymphocytes of ptients with infectious diseses. Infect. Immun. 8: Bentwich Z., nd H. G. Kunkel Specific properties ofhumn B nd T lymphocytes nd ltertions in diseses. Trnsplnt. Rev. 16: Bloom, B. R., L. Jimenez, nd P. I. Mrcus A plque ssy for enumerting ntigen-sensitive cells in delyed-type hypersensitivity. J. Exp. Med. 132: Boyum, A Isoltion of mononucler cells nd grnulocytes from humn blood. Scnd. J. Clin. Lb. Invest. 21(Suppl. 97): DeHortius, R. J., R. G. Stricklnd, nd R. C. Willims, Jr T nd B lymphocytes in cute nd

5 114 THORLEY ET AL. chronic heptitis. Clin. Immunol. Immunopthol. 2: Dougls, R. G., R. H. Alford, T. R. Cte, nd R. B. Couch The leukocyte response during virl respirtory illness in mn. Ann. Intern. Med. 64: Enberg, R. N., B. J. Eberle, nd R. C. Willims, Jr T nd B cells in peripherl blood during infectious mononucleosis. J. Infect. Dis. 130: Gtti, R. A., nd R. A. Good The immunologicl deficiency diseses. Med. Clin. North Am. 54: Hvens, W. P., nd R. E. Mrck The leukocytic response of ptients with experimentlly induced infectious heptitis. Am. J. Med. Sci. 212: Hickling, R. A The monocytes in pneumoni. Arch. Intern. Med. 40: Hung, S.-W., D. B. Lttos, D. B. Nelson, K. Reed, nd R. Hong Antibody-ssocited lymphotoxin in cute infection. J. Clin. Invest. 52: Jimenez, L., B. R. Bloom, M. R. Blume, nd H. F. Oettgen On the number nd nture of ntigensensitive lymphocytes in the blood of delyed-hypersensitive humn donors. J. Exp. Med. 133: Kunkel, H. G Surfce mrkers ofhumn lymphocytes. Johns Hopkins Med. J. 137: Niklsson, P. M., nd R. C. Willims, Jr Studies of peripherl blood T nd B lymphocytes in cute infections. Infect. Immun. 9:1-7. INFECT. IMMUN. 15. Notkins, A. L., S. E. Mergenhgen, nd R. J. Howrd Effect of virus infections on the function of the immune system. Annu. Rev. Microbiol. 24: Piessens, W. F., P. H. Schur, W. C. Moloney, nd W. H. Churchill Lymphocyte surfce immunoglobulins: distribution nd frequency in lympho-prolifertive diseses. N. Engl. J. Med. 288: Sheldon, P. J., M. Ppmichil, E. H. Hemsted, nd E. J. Holborow Thymic origin of typicl lymphoid cells in infectious mononucleosis. Lncet i: Sprunt, T. P., nd F. A. Evns Mononucler leukocytosis in rection to cute infections (infectious mononucleosis). Bull. Johns Hopkins Hosp. 31: Stephens, D. J The occurrence of myelocytes in the peripherl blood in lobr pneumoni. Am. J. Med. Sci. 188: Westmorelnd, D., S. St. Jeor, nd F. Rpp The development by cytomeglovirus-infected cells of binding ffinity for norml humn immunoglobulin. J. Immunol. 116: Winchester, R. J., S. M. Fu, T. Hoffmn, nd H. G. Kunkel IgG on lymphocyte surfces; technicl problems nd the significnce of third cell popultion. J. Immunol. 114: Wybrn, J., nd H. H. Fudenberg How cliniclly useful is T nd B cell quntittion? Ann. Intern. Med. 80: Downloded from on April 17, 2018 by guest