INFECTION AND IMMUNITY, Nov. 1973, p. 700-707Copyright 0 1973 American Society for Microbiology

Vol. 8, No. 5Printed in U.S.A.

Inhibition of Macrophage Migration byNucleotide-Containing Streptococcal

PreparationsB. BULTMANN, B. HEYMER, W. SCHACHENMAYR, 0. HAFERKAMP, AND W. C. SCHMIDT

Department of Pathology, University of Ulm, West Germany, and Department of Pediatrics, ClevelandMetropolitan General Hospital, Case Western Reserve University, Cleveland, Ohio 44109

Received for publication 14 May 1973

Certain extracts of streptococcal cell walls are known to inhibit macrophagemigration in vitro. In this study, we attempted to identify the streptococcalcomponents responsible for this phenomenon. Trypsinized cell walls andcytoplasm from groups A and B streptococci were extracted with hot formamidefollowed by acetone precipitation. Subsequent gel filtration in aqueous solutionsyielded a fraction devoid of C-carbohydrate and containing mostly oligonucleo-tides, apparently derived from streptococcal cytoplasm. This fraction signifi-cantly inhibited the migration of peritoneal exudate cells from rats sensitized togroups A and B streptococci. It was noteworthy that no inhibition of migrationwas observed with cells from nonsensitized animals or control rats injected withBCG or complete Freund adjuvant. Similarly, no inhibition was obtained withformamide extracts of calf thymus RNA. Although the inhibition does not showspecificity for streptococcal groups, it seems to have immunological specificitysince prior sensitization with streptococci is required for migration inhibition.

Extracts of streptococcal cell walls containcomponents which induce loss of peritonealmacrophages when injected into sensitized ani-mals (9, 10). These factors also inhibit macro-phage migration in vitro (migration inhibitionreaction [MIR]) in rats (8). Initial sensitizationof the experimental animals with streptococci isessential-we observed no response in either ofour test systems with nonsensitized controlanimals (8-10). The present study providesevidence that the active components in suchformamide extracts are oligonucleotides ratherthan C-carbohydrate. In addition, we consid-ered it important to ascertain whether theseoligonucleotides are structural components ofthe cell wall or, altematively, cytoplasmic con-taminants of the cell-wall preparations. Theactive component was therefore isolated andchemically characterized, with subsequentcomparison of its biological activity (MIR) andthat of streptococcal cytoplasmic preparations.

Several investigators (16, 28) have demon-strated that nucleic acids and nucleoproteinsstimulate the humoral immune system. Aneffect on cell-mediated hypersensitivity has alsobeen observed: delayed skin reactions are in-duced by bacterial nucleoproteins or nucleicacids (20, 25, 34, 35). Moreover, both homolo-

gous (1) and yeast (31) ribonucleic acid (RNA)modify blast transformation induced by phy-tohemagglutinin in human or animal lympho-cytes. Rejection of mouse-skin allografts is de-layed after administering RNA extracted fromtumors (30), and both the humoral and cellularimmune systems are affected by synthetic poly-nucleotides (5, 23).

MATERIALS AND METHODSPreparation of streptococcal cell walls. The

following strains of streptococci were used for prepar-ing cell-wall and cytoplasmic extracts: group A-strain B 196 (type 17), strain B 930 (type 3), strain T22-76-1 (type 22); group B-strain 090 R. Thesestrains were obtained from the collection of theRockefeller University, N.Y., with the exception ofstrain B 196 (33). All streptococci were grown for 18 hat 37 C in Todd-Hewitt broth (Difco Laboratories,Detroit, Mich.). The cells were harvested, washedtwice with isotonic saline, and stored at -20 C.Cell-wall preparations were made from heat-inac-tivated (30 min at 56 C) groups A and B streptococciby utilizing a Braun disintegrator (Melsungen, Ger-many) according to the method of Bleiweis et al.(4). Differential centrifugation to separate cell compo-nents was then performed by the Freimer (18) proce-dure. Surface proteins were removed by tryptic diges-tion (10), and the deproteinized cell walls were

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subsequently washed three times with isotonic NaCland water and lyophilized. Preparations to be treatedwith ribonuclease (RNase) or deoxyribonuclease(DNase) were not lyophilized but stored at -20 Cuntil used.Treatment of cell-wall preparations with RNase

and DNase. Some of the cell-wall preparations(streptococcal group A, strain B 196, type 17) weretreated with RNase and DNase after digestion withtrypsin. A suspension of cell walls in phosphate-buf-fered saline (PBS) (0.075 M, pH 7.5) was incubated for4 h during constant shaking at 37 C with five-times-crystallized pancreatic RNase (Serva, Heidelberg,Germany) at a concentration of 18.5 Ag of RNase permg of wet cell-wall material. The cell walls were thencollected by centrifugation (30 min, 10,000 x g),resuspended in PBS containing 1 mM MgCl2, andincubated for 16 h at 37 C with 1.85 Mg of bovinepancreatic DNase (Serva) per mg of wet cell-wallpreparation. Finally, the cell walls were washedseveral times with isotonic NaCl and water andlyophilized.

Extraction with formamide and fractionation bygel filtration. Lyophilized cell-wall preparations weretreated with formamide (Merck, Darmstadt, Ger-many) at 170 C for 30 min by the method of Krauseand McCarty (26). A fraction rich in C-carbohydrate(CW-I) was precipitated with acetone from the form-amide extracts (19). This preparation was then di-vided by gel filtration on Sephadex G-25 fine (Phar-macia, Uppsala, Sweden) into two fractions: a car-bohydrate-containing (CW-II) and a carbohydrate-free (CW-III) component (10). CW-III was furtherfractionated on Sephadex G-10 (Pharmacia) into twomore components, CW-IV and CW-V. Water, whichhas been used routinely as eluent, was replaced by 4M urea in some of the experiments. The effluentswere pooled by optical density (OD) at 256 nm, andthe pooled fractions were subsequently lyophilized.

Fractionation of streptococcal cytoplasm. Heat-inactivated groups A and B streptococci were dis-rupted with a Braun disintegrator (4), and cytoplasmwas obtained by differential centrifugation (18). Thecytoplasmic fraction was dialyzed for 24 to 48 hagainst distilled water, and then lyophilized. Form-amide extraction, precipitation with acetone, and gelfiltration on Sephadex G-25 and G-10 were performedas described above.

Extraction ofRNA with formamide. Calf thymusRNA (Serva) was subjected to formamide extractionand acetone precipitation as previously described.The precipitate (TH-RNA) was dissolved in waterand used without further fractionation.Chemical characterization. Cytoplasmic and cell-

wall preparations from streptococci were analyzed forthe following compounds: rhamnose (15), ribose (12),and deoxyribose (11). Organic phosphate was deter-mined by the method of Chen et al. (13). Ultraviolet(UV) spectra were obtained with a Beckman spectro-photometer, model GT (Beckman Instruments, Mu-nich, Germany). For silica gel, thin-layer chromatog-raphy (Silicagel F 254, Merck), the following solventsystem was used: n-butanol, acetone, acetic acid,ammonia, water (7:5:3:3:2). For cellulose thin-layerchromatography (TCL-Cellulose, Merck), methanol,

concentrated HCl, and water (7:2:1) and the abovesolvent system were used. The following bases andtheir nucleoside, deoxynucleoside, and monophos-phoric acid nucleotide derivatives were employed asmarkers: adenine, guanine, inosine, cytidine, thy-mine, and uracil; in addition, we used hypoxanthine,xanthine, and thiouracil (all from Nutritional Bio-chemicals Corp., Cleveland, Ohio). Marker sub-stances in 2-uliter amounts (50 Mmol/ml) were ap-plied to the thin-layer plates side-by-side with ex-tracts from cell-wall preparations and cytoplasm.The plates were developed at room temperature for45 to 175 min and examined by UV light. Oligonu-cleotide-containing preparations were hydrolyzedwith 5.7 N HCl for 1 h at 100 C before analysis of thebase composition.Experimental animals. We used a total of 274

male and female inbred Lewis rats, 4 to 6 months ofage and weighing 200 to 300 g each. Both hindfootpads of 85 rats were subcutaneously injected with0.1 ml of a preparation containing 10 mg of live groupA streptococci (strain B 196, type 17) in completeFreund adjuvant (Difco). All the animals simultane-ously received a second dose of 10 mg of streptococciwithout Freund adjuvant intradermally in the back.Twenty rats were sensitized in the same manner withlive group B streptococci (strain 090 R). Ten moreanimals each received 0.1 ml complete Freund adju-vant, while another ten were injected with BCG inincomplete Freund adjuvant. There were 149 nonin-jected control animals.

Inhibition of macrophage migration. Two weeksafter sensitization, the experimental rats were eachinjected intraperitoneally (i.p.) with 5 ml of Bayol F(Esso, Hamburg, Germany). Four days later, theyreceived 30 ml of Eagle medium (Behring-Werke,Marburg, Germany) i.p. supplemented with 5 U ofheparin (Hoffman-La Roche, Grenzach, Germany),100 IU of penicillin, and 100 Ag of streptomycin per mlof the medium. The inhibition of macrophage migra-tion was examined as described (22); the migrationindex (MI) was expressed as follows: MI (%) =[(average area covered by antigen-containing cul-tures)/(average area covered by antigen-freecultures)] x 100.

Skin tests. Skin tests were performed with five ratssensitized with group A streptococci and five nonsen-sitized control animals, using preparations of strep-tococcal cell walls and cytoplasm (10 to 100 Mg/0.1 mlof saline). Macroscopic inspection as well as histologi-cal methods were employed for evaluation.

Statistical analysis of results. Student's t-testwas used to determine the significance of resultsobtained with the MIR assay and to calculate Pvalues from duplicate values.

RESULTSPreparation and analysis of nucleotide-

containing streptococcal cell-wall extracts.As we had anticipated, an extract of group Astreptococcal cell walls obtained by hydrolysiswith hot formamide and subsequent precipita-tion with acetone (CW-I) (see Table 1) con-tained large quantities of C-carbohydrate as

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evidenced by its rhamnose content (28 to 36%)and reaction with specific antisera to C-car-bohydrate. This extract, however, also con-tained significant amounts of other compo-nents, some of which were dialyzable. Thus gelfiltration on Sephadex G-25 fine resulted in twopeaks (Fig. 1) with components distinct inmolecular weight and in several other proper-ties. The first material (CW-II) eluted from thecolumn exhibited absorption only at 220 nm, itreacted strongly to antiserum against C-car-bohydrate, was nondialyzable, and containedall the group-specific C-carbohydrate of CW-I,judging from the rhamnose content (Table 1).The second peak (CW-III) represented dialyza-ble material, had maximal absorption at 259nm, contained no rhamnose, and did not reactto antiserum against C-carbohydrate. Furtherfractionation of CW-lII was achieved by gelfiltration on Sephadex G-10. As shown in Fig. 2,two peaks were again obtained, with maximalabsorption at -256 nm (CW-IV, CW-V). Boththese fractions were subjected to further analy-sis (Table 2). Organic phosphate, ribose, andtrace amounts of deoxyribose, compoundswhich characterize nucleic acids and nucleo-tide, were detected. CW-IV was about twice asrich in these substances as CW-V. Nucleotideswere demonstrable in fraction CW-IV by thin-layer chromatography. After hydrolysis withHCI, the following purine and pyrimidine baseswere identified: guanine, adenine, uracil, andthymine (Table 2).In some experiments, streptococcal cell-wall

E

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1.5-

1.0-

0.5

preparations were treated with RNase andDNase prior to extraction with formamide andgel filtration. In these experiments, we obtainedonly about 20 to 25% of the amount of oligonu-cleotides found in untreated cell-wall prepara-tions.Preparation and analysis of nucleotide-

containing cytoplasmic extracts. Streptococ-cal cytoplasm was treated with formamide, andthe extract was precipitated with acetone as de-scribed for cell-wall preparations. This extract(CY-I) was fractionated into three componentsby gel filtration on Sephadex G-25 (Fig. 3). Oneof these (CY-II) appeared in the void volume ofthe column, while the other two were found inthe area of low molecular-weight substances(CY-III and CY-IV). We were unsuccessful inattempting to further fractionate CY-HI andCY-IV on Sephadex G-10. CY-III and CY-IVwere both dialyzable, with absorption peaks at256 nm, contained no detectable rhamnose, anddid not react to antiserum against C-carbohy-drate (Table 1). Ribose, deoxyribose, and or-ganic phosphate were detected in CY-IV. Awhole series of nucleotides was disclosed bythin-layer chromatography, and again adenine,guanine, thymine, and uracil were identifiedafter acid hydrolysis (Table 2).Preparation of a calf thymus RNA extract.

Calf thymus RNA was treated with hot form-amide. Subsequent acetone precipitation of theextract yielded a preparation (TH-RNA) withan absorption peak at 256 nm, characteristic ofnucleic acids or bases. This preparation was not

Ivvcw .c II

220nm1|2I I

25l4nm K

I

280nm I

.I.

Sephadex - G 25 finrV V: 55 mlEluent: dist. H20

Fraction No10 20 30 40 50 60Effluent ml 30 60 90 120 150 180PrecipTest --.+ + ++ + _-

FIG. 1. Gel filtration of a formamide extract of group A streptococcal cell-wall preparation (CW-1).Abbreviations: Absorption, E; void volume, VV; CW-II and CW-III, see text; capillary precipitation test withrabbit antiserum against streptococcal C-carbohydrate, Precip. Test.

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TABLE 1. Chemical and immunological characteristics of group A streptococcal cell-wall and cytoplasmicpreparations

Precipitation MaximumPrepn''a | Rhamnose (%) wistreptococcal absorption Dialyzable Mol wtc

antiserum" (2-9 m

Cell-wallCW-I 28-36 + 220 < 10,000CW-II 56-63 + 220 _ -9,000CW-Ill 0 _ 259 + < 5,000CW-IV 0 _ 256 + 5,000 to 700CW-V 0 _ 257 + <700

CytoplasmicCY-IV 0 256 + 5,000 to 700

a See text.b Capillary precipitation test with rabbit antiserum against group A streptococcal C-carbohydrate.c By gel filtration.d Partially dialyzable.

lvv

cwIV cw v

256nm2200rwnm

l~~~~~~~~~~~~~~~~I %

I/

Sephadex -G 10VV: 75 mlEluent: dist. H20

I I

Fraction No. 10 20 30 40 50 60Effluent ml 30 60 90 120 150 180

FIG. 2. Gel filtration of a group A streptococcal C-carbohydrate-free cell-wall fraction (CW-III). Abbrevia-tions: Absorption, E; void volume, VV; CW-IV and CW-V, see text.

TABLE 2. Nucleic acid components of group Astreptococcal cell-wall and cytoplasmic preparations

obtained by gel filtration

aOrganic Ribose Deoxy-Prepn phosphate (jig/mg), ribose Basesc

(jg/mg), (aUg/mg)"

CW-IV 20.8 38.2 7.9 GU, AD,UR, THY

CW-V 8.1 15.3 4.8 Not doneCY-IV 33.5 104 14.4 GU, AD,

I___I____ I___ I____ UR, THYa See text.'Per milligram of preparation.c GU, Guanine; AD, adenine; UR, uracil; THY,

thymine.

analyzed in detail but was used without furtherfractionation in later biological assays.

Effect of various preparations on macro-phage migration. Crude extracts of group Astreptococcal cell-wall preparations (CW-I) inconcentrations of 50 ,ug/ml medium caused astatistically significant inhibition of macro-phage migration in peritoneal exudate cellsfrom animals sensitized with group A strepto-cocci (Fig. 4). No effect was apparent with cellsfrom nonsensitized control animals. Pure C-car-bohydrate (CW-II) isolated by gel filtrationcaused no inhibition of migration in cells fromsensitized or nonsensitized rats; on the con-trary, in some experiments, we observed a slight

E

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Ivy

CY 11, ,CY 111,,YU IV Sephodex -G 25 fineVV: 55 mlEluent: dist.H20

220 nmi

I \I \

'.II

Froction No. 10 20 30 L0 50 60Effluent ml 30 60 90 120 150 180

FIG. 3. Gel filtration of a formamide extract ofgroup A streptococcal cytoplasm. Abbreviations: Absorption,E; void volume, VV; CY-II, CY-III, and CY-IV, see text.

enimelNo. 25 27 x tt10 1 10 101 4 57-13N/S N S N S N S |N S N S N S

P NS >0.001 NS |NS NSD05|ODS 0005 >0.2aO2 NS>0001preporotiof CW I CW IIT CW III IVl cw v CY IV

pig/irA 50 50 25 T2s55 25 25

FIG. 4. MIR assay of various streptococcal group A cell-wall fractions and cytoplasmic extracts. Abbrevia-

tions: N, nonsensitized animals; S, animals sensitized with group A streptococci; P, P values by Student's

t-test; NS, differences statistically insignificant; CW-I, II, III, IV, V, and CY-IV, see text.

increase in cell motility. However, a stronginhibition of macrophage migration was notedwith the nucleotide-containing C-carbohydrate-free fractions CW-III and CW-IV (Fig. 4). Theinhibition of migration was correlated with thecontent of oligonucleotides. The preparationwith the highest content of oligonucleotides(CW-IV) inhibited migration at lower concen-trations (5 sg/ml), whereas those containinglesser concentrations of oligonucleotides re-

quired higher concentrations (e.g., 25 jg/ml[CW-III] and 50 ,g/ml [CW-I]). Fraction CW-Vwas less potent than the other fractions and alsodiffered in that it inhibited migration of cellsfrom both sensitized and nonsensitized rats to asimilar degree (Fig. 4).

Cytoplasmic preparations CY-III and CY-IVfrom both group A and B streptococci in con-centrations of 25 Ag/ml medium caused statisti-cally significanA inhibition of migration of peri-

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toneal exudate cells from rats sensitized withgroup A streptococci; no effect was observedwith cells from nonsensitized animals. Althoughfraction CY-IV had been obtained from group Astreptococci, it had a similar inhibitory effecton the migration of macrophages from animalssensitized with either group A or group B strep-tococci. The same results were obtained whengroup B streptococcal fraction CY-IV was em-ployed.Control experiments. The oligonucleotide

fraction CW-IV (25 usg/ml medium) obtainedfrom streptococcal cell-wall preparations didnot inhibit the migration of macrophages fromrats sensitized with BCG or complete Freundadjuvant alone. In addition, oligonucleotidepreparations (10 to 15 ,g/ml medium) obtainedby formamide extraction of calf thymus RNAdid not inhibit migration in either cells fromanimals sensitized with streptococci or thosefrom untreated control animals.The active substances in the MIR assay were

completely dialyzable; no activity was left inthe nondialyzable material after 24-h dialysis ofcell-wall fraction CW-Ill against Eagle mediumat 4 C. When C-carbohydrate-free cell-wall ex-tracts or fractions active in the MIR assay weresubjected to Sephadex G-10 filtration in 4 Murea, elution profiles identical to that shown inFig. 2 emerged. We estimated the molecularweight of the oligonucleotides of the C-carbohy-drate-free extracts to be 700 to 5,000 on thebasis of their elution pattems on Sephadex G-10and G-25 in 4 M urea or distilled water.

DISCUSSIONEarlier studies have established that formam-

ide extracts of trypsinized cell-wall prepara-tions obtained from group A streptococci inhibitthe migration of peritoneal macrophages fromanimals previously sensitized with streptococci.The present investigation demonstrates thatthis reaction is not due to the group-specificC-carbohydrate (CW-II) which is the maincomponent of such cell-wall extracts. By usinggel filtration, we obtained a preparation devoidof C-carbohydrate (CW-IV) with a 10-fold in-crease in migration inhibition activity as com-pared to that of the starting material (CW-I).Migration inhibition was observed only withperitoneal macrophages from animals sensi-tized with group A or B streptococci, not withcells from untreated rats. It was noteworthythat prior sensitization with streptococci wasnecessary, but there was no group specificity.The active component had an absorption peakat 256 to 259 nm, which is characteristic forbases of nucleic acids. Ribose, organic phos-

phate, and trace amounts of deoxyribose weredetected. After hydrolysis with HCl, guanine,adenine, thymine, and uracil were identified inthe preparations by thin-layer chromatography.These results indicate that the active principlein the inhibition of migration in this system isoligonucleotide. Preparations containing eithertrace amounts of peptides or none were equallyactive in inhibiting macrophage migration.

After identifying the MIR-active substancesas oligonucleotides, we tried to determinewhether these oligonucleotides are intrinsiccomponents of streptococcal cell walls or merelycytoplasmic contaminants of the cell-wall prep-arations. Barkulis and Jones (2) described theoccurrence of nucleic acids in streptococcalcell-wall preparations and demonstrated thatthey derived from absorption onto cell wallsduring the isolation procedure. Salton (32) sug-gested that their presence might be explainedby cytoplasmic contamination. Nevertheless,he found a uridine-diphosphonucleotide boundto N-acetylmuramic acid in group A streptococ-cal cell walls, and Park (27) observed accumula-tion of uridine-5'-diphospho-N-acetyl-aminosugars in staphylococcal cell walls after expo-sure of cells to certain antibiotics. However, themost cogent evidence that the MIR-producingnucleotide fraction described here is of cytoplas-mic origin is indicated by the fact that formam-ide extracts of streptococcal cytoplasm yieldedoligonucleotides similar in chemical composi-tion, molecular size, and MIR activity to thosein extracts from partially purified cell walls.

Relatively little information on the biologicalactivities of oligonucleotides is available. Ac-cording to Freedman and Braun (17), oligonu-cleotides activate the reticuloendothelial sys-tem. Braun et al. (6, 7) and Johnson et al. (24)also reported nonspecific stimulation of primaryand secondary immune responses by these com-pounds. Aside from eliciting such adjuvant-likeeffects, oligonucleotides can act as antigens orhaptens, and antibodies against them can beinduced if coupled to suitable carriers (3, 16, 28,29).At present, it is not clear whether oligonucleo-

tides are also active in the cellular immunesystem. Stimulation of lymphocytes by syn-thetic polynucleotides has been observed usingcertain strains of mice; however, Van Boxel etal. (36) could produce no such effect in sensi-tized guinea pigs, nor did they obtain positiveskin reactions with polynucleotides. The MIRtest used in the present investigation is consid-ered to be an in vitro assay for cellular hyper-sensitivity (14), although other activities un-related to immune reactions may also be as-sayed by this method (21, 22). The observation

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that only the peritoneal macrophages of ani-mals sensitized by streptococci reveal migrationinhibition would appear to indicate some im-mune specificity in the streptococcal oligonu-cleotides used in this study, since macrophagesof control animals or those injected only withBCG or Freund adjuvant did not respond. Norwas there any reaction with formamide extractsof calf thymus RNA, using either sensitized orcontrol animals. The sera of sensitized ratscontained low titers of humoral antibodiesagainst streptococcal mucopeptides and C-car-bohydrates but not against streptococcal nu-cleic acids (unpublished observations). How-ever, delayed skin reactions could not be in-duced with our oligonucleotide preparations ineither sensitized or untreated animals. Thismight be explained by the very rapid break-down of oligonucleotides by enzymes after injec-tions (7). Further characterization of the im-mune responses to streptococcal oligonucleotidepreparations will help to resolve the question ofwhether inhibition of macrophage migrationcan be considered a specific immune reaction orwhether some other biological effects of oligonu-cleotides are involved.

ACKNOWLEDGMENTS

This work was supported by Deutsche Forschungsgemein-schaft (Bui 293/1). The excellent technical assistance ofBeatrice Schibli and Kirsten Wachholz is gratefully acknowl-edged.

LITERATURE CITED

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2. Barkulis, S. S., and M. F. Jones. 1957. Studies ofstreptococcal cell walls. I. Isolation, chemical composi-tion, and preparation of M-protein. J. Bacteriol.74:207-216.

3. Beiser, S. M., V. P. Butler, and B. F. Erlanger. 1968.Hapten-protein conjugates: methodology and applica-tion, p. 15-24. In P. A. Miescher and H. J. Muller-Eberhard (ed.), Textbook of immunopathology. Grune& Stratton, New York.

4. Bleiweis, A. G., W. W. Karakawa, and R. M. Krause.1964. Improved technique for the preparation of strep-tococcal cell walls. J. Bacteriol. 88:1198-1200.

5. Braun, W., M. Ishizuka, Y. Yajima, D. Webb, and R.Windechurch. 1971. Spectrum a mode of action of polyA: U in the stimulation of immune responses, p.139-156. In R. F. Beers and W. Braun (ed.), Biologicaleffects of polynucleotides. Springer-Verlag, Berlin.

6. Braun, W., and M. Nakano. 1965. Influence of oligodeox-yribonucleotides on early events in antibody formation.Proc. Soc. Exp. Biol. Med. 119:701-707.

7. Braun, W., M. Nakano, L. Jaraskova, Y. Yajima, and L.Jimenez. 1968. Stimulation of antibody-forming cellsby oligonucleotides of known composition, p. 347-363.In 0. J. Plescia and W. Braun (ed.), Nucleic acids inimmunology. Springer-Verlag, Berlin.

8. Bfiltmann, B., B. Heymer, 0. Haferkamp, and W. C.Schmidt. 1970. Untersuchungen zur zellgebundenenUberempfindlichkeit gegen Streptokokkenzellwandan-

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kamp. 1971. Peritoneal macrophage disappearance re-action in the rat. Z. Immunitaetsforsch. Allerg. Klin.Immunol. 142:267-275.

10. Biiltmann, B., C. P. Sodomann, B. Heymer, 0. Hafer-kamp, and W. C. Schmidt. 1971. Loss of macrophagesfrom peritoneal exudates of sensitized rats after injec-tion of streptococcal antigens. Int. Arch. Allergy Appl.Immunol. 41:491-500.

11. Burton, K. 1956. A study of the conditions and mech-anisms of the diphenylamine reaction for the colorimet-ric estimation of deoxyribonucleic acid. Biochemistry62:315-323.

12. Ceriotti, G. 1965. Determination of nucleic acids inanimal tissues. J. Biol. Chem. 214:59-70.

13. Chen, P. S., T. Y. Toribara, and H. Warner. 1956.Microdetermination of phosphorus. Anal. Chem.28:1756-1759.

14. David, J. R. 1971. Migration inhibitory factor and media-tors of cellular hypersensitivity in vitro, p. 399-412. InB. Amos (ed.), Progress in immunology. AcademicPress Inc., New York.

15. Dische, Z., and L. R. Shettles. 1948. A specific colorreaction of methylpentoses and a spectrophotometricmicromethod for their determination. J. Biol. Chem.175:595-603.

16. Erlanger, B. F., and S. M. Beiser. 1964. Antibodiesspecific for ribonucleosides and ribonucleotides andtheir reaction with DNA. Proc. Nat. Acad. Sci. U.S.A.52:68-74.

17. Freedman, H. H., and W. Braun. 1965. Influence ofoligodeoxynucleotides on phagocytic activity of thereticuloendothelial system. Proc. Soc. Exp. Biol. Med.120:222-225.

18. Freimer, E. H. 1963. Studies of L-forms and protoplastsof group A streptococci. II. Chemical and immunologi-cal properties of the cell membrane. J. Exp. Med.117:377-399.

19. Fuller, A. T. 1938. The formamide method for theextraction of polysaccharides from hemolytic strepto-cocci. Brit. J. Exp. Pathol. 19:130-139.

20. Hana, I., M. Koleckarova, and L. Klenka. 1968. Cutane-ous delayed-type reactions to different components ofgroup A streptococcus in patients with recurrent eyediseases and in normal controls. Acta Allergol.23:374-386.

21. Heilman, D., and R. C. Bast. 1967. In vitro assay ofendotoxin by the inhibition of macrophage migration.J. Bacteriol. 93:15-20.

22. Heymer, B., B. Biultmann, and 0. Haferkamp. 1971.Toxicity of streptococcal mucopeptides in vivo and invitro. J. Immunol. 106:858-861.

23. Johnson, A. G., R. E. Cone, H. M. Friedman, I. H. Han,H. G. Johnson, J. R. Schmidtke, and R. D. Stout. 1971.Stimulation of the immune system by homopolyribo-nucleotides, p. 157-177. In R. F. Beers and W. Braun(ed.), Biological effects of polynucleotides.Springer-Verlag, Berlin.

24. Johnson, A. G., J. R. Schmidtke, K. Merrit, and I. H.Han. 1968. Enhancement of antibody formation bynucleic acids and their derivatives, p. 379-385. In 0. J.Plescia and W. Braun (ed.), Nucleic acids in immunol-ogy. Springer-Verlag, Berlin.

25. Kahn, M. C., H. S. Baldwin, B. R. Zeitlin, and M. Smart.1951. Cutaneous reactions to staphylococcus polysac-charide, proteins, and an unfractionated extract inhypersensitive and normal individuals. J. Allergy22:237-248.

26. Krause, R., and M. McCarty. 1961. Studies on thechemical structure of the streptococcal cell wall. I. Theidentification of a mucopeptide in the cell wall of groupA and A-variant streptococci. J. Exp. Med.114:127-140.

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