CYTOLOGICAL STUDIES IN ASSOCIATIONWITH LOCAL INJECTIONS OFSTREPTOKINASE-STREPTODORNASE INTOPATIENTS

Alan J. Johnson

J Clin Invest. 1950;29(10):1376-1386. https://doi.org/10.1172/JCI102375.

Research Article

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CYTOLOGICALSTUDIES IN ASSOCIATION WITH LOCAL IN-JECTIONS OF STREPTOKINASE-STREPTODORNASE

INTO PATIENTS 1

By ALAN J. JOHNSON

(From The Department of Medicine, New York University College of Medicine, and the ThirdMedical Division, Bellevue Hospital, New York City)

(Submitted for publication May 6, 1950; accepted, July 18, 1950)

The use of streptokinase 2 and streptococcaldesoxyribonuclease 2 as therapeutic agents hasbeen the subject of recent reports from this clinicand laboratory (1, 2, 3). The injection of theseenzymes into areas of inflammatory exudation hasbeen observed (1) to be accompanied by a localoutpouring of leukocytes. A striking decrease inthe~ number of degenerated leukocytes as well asa rapid disappearance of extracellular desoxyribosenucleoprotein has also been described. Thesechanges have been attributed to the action of thestreptococcal desoxyribonuclease (2).

In order to obtain a more complete under-standing of the effects of these lytic enzymes, thecytological and cytochemical reactions evokedlocally have been studied in some detail, togetherwith direct observations of the viability, motilityand phagocytic activity of leukocytes both beforeand subsequent to enzymatic therapy.

The studies to be described in this article maybe divided into the following categories:

1) Effect of injections of SK-SD on the totalnumber, stpravital staining, and motility of whiteblood cells in pleural exudates of patients.

2) Differential counts of exudates followinginjections of SK-SD in patients.

3) In vitro studies of phagocytic activity ofleukocytes following injection of SK-SD in pa-tients.

4) Relationship of extracellular desoxyribosenucleoprotein to clumping of leukocytes in exu-dates.

5) Effect of SK-SD on the staining of cellnuclei of viable and degenerated cells in exudatesas determined by the Feulgen reaction.

I This study was supported in part by a grant from theNational Institutes of Health, U. S. Public Health Service.

2 For brevity, Streptokinase is referred to as SK andstreptococcal desoxyribonuclease as SD. SK-SD indi-cates the mixture.

6) Correlation of enzymatic depolymerizationas determined biochemically with cellular changesin pleural exudates of patients.

MATERIALS AND METHODS

I. Streptococcal concentrate. SK-SD3 was con-tained in crude and partially purified concentrates pre-pared and measured by methods described by Christensen(4). In most instances the preparations contained bothenzymes in high concentrations. Occasionally, a concen-trate was used that had a very high SK titer and a verylow SD titer. Although it would have been desirable toemploy preparations containing SK or SD alone, at thetime this work was in progress the only preparations avail-able for therapeutic use contained both enzymes. Fur-ther studies with purified SK and SD, respectively, are be-ing carried out. Special mention will be made of enzymetiters in individual protocols.

All enzyme preparations employed for studies in vzitrowere dialyzed against Simm's buffer 4 at 40 C forthree days in order to attain a physiologically balancedsolution with an osmotic pressure comparable to thatof human serum. The osmotic pressures were then calcu-lated from cryoscopic determinations. The freezingpoints had a range of - 0.55° to - 0.58° C. No preserva-tive was used in these preparations. Sodium bicarbonateand phosphate buffers were employed exclusively in theinitial preparation and purification of the enzymes.

The crude material contained about 16-20 units of SK(4) and 3-4 units of SD (4) per gammaof nitrogen andwas found to be relatively stable in a concentrated solu-tion in the cold. The protamine purified preparationscontained about 150-200 units of SK, about 40-200 units ofSD per gamma of nitrogen and were relatively unstableover long periods of time unless a non-specific proteinwas added as a protective colloid. In the case of thecrude lyophilized enzyme, stability was attained by us-ing a final concentration of about 2% of the crude enzyme,and in the case of the protamine purified, it was assuredby adding human serum, that had been shown to havelow anti-streptokinase and anti-streptodornase titers, toa final concentration of 0.2%o to 2%o.

13A number of the preparations were supplied byLederle Laboratories.

4 The formula of Simm's buffer is the same as Simm'sPhysiological Solution "X7" manufactured by Microbio-logical Associates.

1376

CYTOLOGICAL STUDIES WITH LOCAL INJECTIONS OF SK-SD

All studies in vitro were performed with duplicate ali-quots. The controls were set up with the same enzymesolutions which had been previously incubated at 56° Cuntil enzyme activity was no longer demonstrable.

II. Cell counts. Total counts and white blood cellcounts were done in duplicate by standard methods, ex-cept that thick, highly cellular exudates were dilutedin saline to bring the cell numbers within the range ofthe methods. Dilution with saline also served to effecta partial depolymerization and dissolution of the highlyviscous, extracellular desoxyribonucleic acid (DNA)usually present. The white cells tended to clump mark-edly in the presence of the latter, unless primary dilu-tion was effected. Tenth normal HC1 with addedLoeffler's methylene blue was used as the final dilutingfluid for white blood cell counts.

III. Supratntal staining and motility of leukocytesA. Suprazital stain. Supravital staining was per-

formed according to standard techniques (5, 6). NationalAniline Neutral Red, certification No. NX14, was usedin varying concentrations depending on the number ofcells in the fluid studied. The cover-slip technic wasused with silicone stop-cock grease (Dow Corning) in-stead of vaseline. Thick, highly cellular exudates werediluted with Simm's solution, 1: 1 to 1: 5, when neces-sary, to facilitate observation and promote an optimalconcentration of cells for efficient, non-toxic staining.By varying the dilution of the cells and the concentrationof neutral red, the proportion of stain to cell numberswas readily adjusted in spite of the wide variety of exu-dates tested.

Two hundred to 600 cells from the exudates of patientswere observed in duplicate wet preparations on a warmstage (37.5° C) under oil immersion. A mechanicalstage was utilized to c6unt the cells in a standard Z pat-tern on the slide. The per cent staining supravitally wascalculated, and the number staining supravitally was inturn calculated from the percentage value obtained aboveand the total white blood cell count. At least two ob-servations were made within two hours, although manysamples observed for considerably longer time intervalswere found to check with the original data.

B. Motility. Enumeration of the cells exhibiting mo-tility was performed at the same time and under thesame conditions as was the enumeration of cells takingsupravital stain. Motility was expressed as the per centof those cells which took the supravital stain that showeddefinite pseudopods and/or pseudopod formation withprotoplasmic streaming. The actual number of motilecells was calculated from the percentage value obtainedabove, and the number of white blood cells stainingsupravitally.

IV. Phagocytosis. Experiments on phagocytosis werecarried out with a strain of rough pneumococcus, R36N/C. The bacteria were washed three times in neo-peptone broth and diluted in the broth to concentra-tions of 108 to 108 bacteria per milliliter. Nine-tenths ml.samples of exudate were added to 0.1 ml. of washedpneumococci in each of two test tubes and the tubes

were rotated at 15 r.p.m. (7, 8) in a waterbath at 37.5° Cfor 30 to 60 minutes, respectively. The mixture was thensmeared on a slide, stained with Wright's stain, and300-500 white blood cells were observed for ingestedbacteria (9). The highest per cent of leukocytes show-ing phagocytosis was taken as the "phagocytic activity"of that exudate regardless of the concentration of theorganisms. In most cases, maximum phagocytosis wasattained at one hour at a concentration of 10' bacteriaper milliliter. Although there was an excess of bacteria,the number of bacteria per leukocyte was not used asan index because the ratio of bacteria to viable leuko-cytes in the original mixture could not be rigidly con-trolled. In those experiments in which active streptococ-cal desoxyribonuclease was present, depolymerization ofthe DNA in the cell nuclei occurred after traumaticrupture of some of the cells during the process of smear-ing the exudate. This was prevented by the addition ofsodium citrate to a final concentration of 4% just beforesmearing the slides.

V. Desoxyribose nucleic acid (DNA) 5A. Feulgeni stain. Thick and thin smears of exudates

were air dried and Feulgen stained as described in a pre-vious report (2). No fixative was used. Fast Green FCFwas used as a counterstain.

B. Extraction. The polymerized DNAin the sedimentand cells was washed with cold, citrated saline, and ex-tracted with 5% trichloracetic acid (10).

Analysis of the partially depolymerized DNA in thesupernatants was far more difficult because of the pres-ence of interfering substances such as hexoses and degra-dation products from protein hydrolysis following hottrichloracetic extraction. Preliminary results with amethod of extraction utilizing crystalline pancreatic de-soxyribonuclease 6 in 5% bovine albumin have shownthat removal of interfering protein and extraction of DNAwere relatively complete in this method. Treatment ofthe sample under standard conditions was followed byprecipitation of the protein with 5% trichloracetic acid atroom temperature. The acid soluble DNAin the filtrateswas then measured by chemical methods.

C. Analysis. A modification (11) of Dische's di-phenylamine reaction (12), utilizing the difference inabsorption at 6,100 A and 6,500 A, was used for DNAanalyses. Because protein may interfere in the reaction(13, 14) a modification of the cysteine method of Stumpf(15) was employed as a check. The modification usedconsisted of varying the amount of sample from 0.5 ml.to 1.5 ml., and the concentration of sulfuric acid from70% to 90%o (in order to maintain a final concentrationof 63%). A uniform lot of "aged" sulfuric acid solu-

5 Inasmuch as DNA was the substance specificallytested for in exudates froni patients, a sharp distinctionwill not be drawn between desoxyribose nucleoproteinand DNA; and desoxyribose nucleoprotein will usuallybe referred to as DNA.

6 Obtained from the Worthington Laboratories, NewJersey.

1377

ALAN J. JOHNSON

tion was used and separate curves calibrated for eachconcentration.7

The temperature of the reaction was kept below 400 C.When 80% or 90% sulfuric acid was used, the reagentswere iced and sulfuric acid was added to the samples veryslowly in an ice bath. The analyses were performed inBeckman DU and Model B spectrophotometers usingDNAstandards of the sodium salt of thymonucleic acidprepared in this laboratory (N/P ratio 1.6). The modi-fication described increased the sensitivity of the originalmethod threefold.

VI. Other biochemical determinationsA. Phosphorus. Total acid soluble phosphorus, inor-

ganic phosphorus, and organic acid soluble phosphoruswere determined by methods previously described (2).

B. Nitrogen. Acid soluble nitrogen was determinedon 8% trichloracetic acid filtrates by micro-Kjeldahl andNesslerization.

VII. Collection of samples of exudate from patients.No anti-coagulant was used or found to be necessary inthe purulent exudates. Heparin was used routinely innon-'urulent exudates. Since high concentrations ofheparin interfere with phagocytosis and motility (16),the concentration used did not exceed approximately 10mgms. %. Moreover, at this concentration, little or noinhibition of SD occurs.

RESULTS

1) Effect of injections of SK-SD on the totalnumber, supravital staining, and motility ofwhite blood cells in pleural exudates of patients

The local pleural- cellular response to injectionsof SK-SD has been observed in patients withnon-purulent, serous effusions resulting frombronchogenic carcinoma, cardiovascular-renal dis-ease, and tuberculous pleurisy.8 Enzyme con-centrates with combined unit dosages rangingfrom 20,000 SK-4,000 SD to 100,000 SK-25,000SD contained in 5-10 ml. of diluent were em-ployed and total WBCcounts were made beforeand 24 hours after the injection.

The results may be summarized as follows:the average of the total number of WBC's beforeinjection was 827 per cu. mm. Twenty-fourhours after injection there were 11,380 WBC's

7 For reasons that are not understood, full color devel-opment and uniform results were not obtained unless"aged" sulfuric acid solution and a standard time interval(60 seconds) for adding the acid to the sample were used.The sulfuric acid solution was heated to 560 C for 12hours, then allowed to remain undisturbed for severalmonths.

sThese observations have been made over a periodof several years by different observers.

per cu. mm. The smallest increase was from1,650 to 2,750 cells per cu. mm.; the greatest wasfrom 400 to 26,800 cells per cu. mm. The changewas largely accounted for by an increase in poly-morphonuclear leukocytes. When two succeed-ing injections were given to the same patient inthis preliminary group within three to five daysafter the effects of the first had subsided, therewas a six to eight times greater outpouring of cellsfollowing the second injection.

In two instances, enzymes inactivated by heat-ing for 24 hours at 560 C were injected in pa-tients with pleural effusions. In one case, thenumber of cells pre- and 24 hours post-treatmentwere 1,100 and remained unchanged. In theother, the number of cells pre- and 24 hours post-treatment were 75 and 180, respectively. It is of

,interest that a significant cellular increase oc-curred later in these patients when they weretreated with an active enzyme preparation of thesame lot as the inactivated material. These re-sults were confirmed in two other instances, whenthe SK-SD was applied locally to a clean, granu-lating ulcer, and a special bandage utilized to poolthe enzyme and collect the exudate. These limitedobservations suggest that the degree of local cel-lular reaction is specifically associated with ac-tivity of SK-SD. The increase did not seem tocorrelate well with enzyme dosage or pyrogenicreactions. Further studies of the several factorscontained in the streptococcal concentrates thatspecifically influence the degree and type of reac-tion are in progress.

Eleven patients with empyema twith thick, puru-lent exudations received 19 injections of SK-SDin varying amounts in the successful therapeuticrange. There were four cases of pneumococcalempyema (two infected, two sterile), three tuber-culous, one due to anaerobic streptococcus, one toB. pyocyaneus, and two sterile empyemas of unde-termined etiology. The dosage of streptokinaseper injection varied from 200,000 to 400,000 units,and that of streptodornase from 5,000 to 200,000units. When a patient received more than oneinjection, the time interval between injections wasdetermined by clinical indications and varied fromtwo days to two weeks. Specimens were pro-cured before injection and one hour or more post-injection during the first day and usually at 24hour intervals thereafter for the duration of the

1378

CYTOLOGICAL STUDIES WITH LOCAL INJECTIONS OF SK-SD

observations. Drainage of the pleural fluid wasnot effected until 24 hours after the injection.Appropriate biochemical and cytological studiesshowed that specific enzyme action had takenplace in each case (1, 2).

The cytological effects of injections of SK-SDin these empyemata are shown in Figure 1.

From Figure 1 it may be noted that there was awide range in the total number of WBC's in thepleural exudate before therapy, varying from4,000 to 500,000 cells per cu. mm. Serial countsfollowing the introduction of the enzymes indi-cated the initial response usually became evident

during the first hour post-treatment and reachedits maximum at 24 hours. In two instances thecount was lower after 24 hours (patients McM,first injection, and BAT, second injection. Al-though not recorded in Figure 1, the maximumnumber of WBC's was usually maintained for 24hours and diminished during the ensuing week toor below the pre-injection level. Many factorspertaining to the underlying disease and degree ofresponse to treatment controlled the number ofcells present during the post-injection period.Under optimal clinical conditions, it was not un-usual for the local WBCcount to fall markedly

600,000

500 ooo - TOTAL NUMBEROF WHITE BLOOD CELLS

ioo,ooo ~PER CU. MM. E

HEN REV MCm BAT HEI KUL CEP LEN BUR BR MACI-2 1-2-3 1- 2 1- 2- 3 I-2 1 1-2 1

350.000

300,000

250,000 C MOTILE WHITE BLOODCELLS(3)

200.000

150,000

100,000

50,000

MEN REV McM BAT HEI KUL CEP LEN BUR O MACI - 2 1 - 2 - 3 1 - 2 1 - 2 -3 - 2 l ,- 2 , , ,

FIG. 1. EFFECTOF INJECTIONS OF SK-SD ONTHE ToTAL NUMBER,SuPRA-VITAL STAINING, AND MOTILITY OF WHITE BLOODCELLS IN PLEURAL Exu-DATESOF PATIENTS

The solid bar represents the number of white blood cells before injectionand the cross-hatched bar, the number of white blood cells 24 hours afterinjection.

When a patient received more than one injection, this is denoted by thenumber under his initials.

1379

ALAN J. JOHNSON

in three to five days. A case demonstrating thispoint is described in a later section.

Plven though the number of white blood cellsper cu. mm. varied inversely with its dilution bypleural fluid, the increase in total number ofWBC's occurred in spite of the increase in pleuralfluid usually observed following the injection ofthe SK-SD.

It was not clearly established, however, whetherthe accumulating cells emerged from the site ofthe inflammation within the tissues or were re-leased from deposits of exudate undergoing lysisby the enzymes. Since a considerable number ofcells in the original, highly viscous exudate weredead or degenerated, the studies on vital stainingstrongly suggest that a major portion of the in-crease in white blood cells was due to an out-pouring of fresh viable cells.

Associated with the change in the total numberof white blood cells, the number of cells stainingsupravitally and those exhibiting motility in-creased in a significant and striking manner whichwas emphasized by the increase in per cent ofcells taking supravital stain and exhibiting mo-tility. These phenomena were consistently notedafter each injection with one exception. Forthe 19 injections, the mean increase in white bloodcells per injection per 24 hours was 112,000 witha standard deviation of 71,400, and the mean in-crease in motile white blood cells was 72,400 witha standard deviation of 67,900. Thus, the in-crease in cells staining supravitally constituted83% of the increase in total WBC's, and the in-crease in motile cells constituted 77% of the cellsstaining supravitally.

Further analysis of the data in Figure 1 showsthat the number of cells staining supravitally andexhibiting motility had undergone a much greaterproportionate increase than the total white count.The ratio of the mean of the post-injection countsto the initial counts indicates that the total whiteblood cell counts increased about twofold, thesupravital staining about fourfold, and the motil-ity about sixfold in 24 hours. For example, inthe cases of BRO, and MAC(Figure 1), the finalor post-injection white count is less than 1.5 timesthe pre-injection white blood cell count whereasthe cells staining supravitally and exhibiting mo-tility increased after injection to more than fourtimes the pre-injection level. In addition, when

the per cent of the total cells staining supravitallywas noted for a number of consecutive time in-tervals following an injection, there was usually a30-60%o rise in supravital staining in 24 hours.If two or more injections were given to the samepatient at one to two week intervals, the responsefollowing the second injection was about twicethat following the first. It would seem, therefore,that the cytological formed elements in these puru-lent pleural fluids undergo a nearly complete"turnover" following injection of the SK-SDfrom a static state with a preponderance of dead,degenerated cells to a dynamic state with a ma-jority of fresh, viable cells.

2) Differential counts of exudates followzing in-jections of SK-SD in patients

The differential counts of exudates from pa-tients with a variety of diagnoses have had onecommon denominator following therapeutic useof the concentrate. No matter what the initial or

I.-zo 50

a:0.

c; 40

0

00 30IQ.

O°t 3 6 24INJECTION TIME, HOURS

FIG. 2. IN VITRO PHAGOCYTIC ACTIVITY OF LEUKO-CYTES IN PLEURAL EXUDATES REMOVEDAT VARYINGINTERVALS AFTER THE LOCAL INJECTION OF SK-SD INPATIENTS

The ordinate represents the per cent of cells showingphagocytic activity.

1380

CYTOLOGICAL STUDIES WITH LOCAL INJECTIONS OF SK-SD

final quantitation, regardless of the ratios of theinitial cell types, 24 hours after the injection thecells were largely those of the polymorphonuclearleukocytic series. Where elimination of infectionor marked improvement occurred following theinjections, the polymorphonuclear series was re-

placed by mononuclear cells of various kinds, a

few lymphocytes at first, then monocytes, macro-

phages and endothelial cells. It was not under-stood what part, if any, the macrophages played.They were rarely present until long after theacute reaction had subsided.

3) In vitro studies of phagocytic activity of leuko-cytes follo7wing injection of SK-SD in patients

Following the injection of SK-SD into thepleural cavities of patients, a significant rise inthe proportion of leukocytes containing ingestedR pneumococci was demonstrable in each of theseven cases presented in Figure 2, as contrastedwith the pre-injection specimen. In the cases offrank empyema, three tuberculous, one pneumo-

coccal, and one sterile empyema of underterminedetiology, the phagocytic activity 24 hours afterfirst therapy was increased by 10-28%. Itwill be recalled that the per cent of cells stainingsupravitally in these cases was considerablyhigher. The other two cases, hydrothorax andhemothorax, respectively, showed a much higherelevation of the per cent of phagocytic activity inthe presence of SK-SD, nearly comparable tothat obtained in vitro in normal whole blood,with or without SK-SD. These findings, withthe low or absent glucose levels in the chest fluidsfollowing injection, suggest that the phagocyticability of the leukocyte may approach a maximumlimited by its nutritional milieu when the exuda-tive coating is thick and the total cell populationrelatively large (17, 18).

Since the purulent exudates from these patientswith empyema usually contained 100,000-200,000white blood cells per cu. mm. while the exudatesfrom the cases of hydrothorax and hemothoraxcontained 1,000-10,000 WBC's per cu. mm.,the total number of cells available for phagocyticactivity was considerable in the cases of empyema.

4) Relationship of extracellular desoxyribonucleicacid to clumping of leukocytes in exudates

The white blood cells of purulent exudates, par-

ticularly in empyema of long standing, were

clumped in masses of varying sizes which werebroken up when SK-SD was introduced (1).Although several thousand slides were reviewed,gross clumping of the white blood cells in puru-lent chest fluids was not observed in the absenceof extracellular DNA. DNA, rather than fibrin,was particularly significant in the clumping phe-nomenon, since the cellular aggregations wereusually seen in cases of thick, purulent empyema,which usually had a high DNAand a low fibrino-gen content. They have not been seen in effu-sions or hemothorax where both the extracellularDNAand the fibrinogen were low. The clump-ing of WBC's in chronic purulent infections wasprobably associated with the high viscosity of theextracellular DNA and was broken up by thestreptodornase. This view was also supportedby the failure of preparations containing hightiters of streptokinase with very low strepto-dornase content to depolymerize the DNA orbreak up the clumps of white blood cells in puru-lent exudates.

5) Effect of SK-SD on the staining of the cellnuclei of viable and degenerated cells in exu-dates as determined by the Feulgen reaction

When fixed tissue, sections and smears weretreated with pancreatic desoxyribonuclease (19,20, 21), the nuclei of the treated cells no longerstained with the Feulgen stain even though thetime of exposure was only five to 10 minutes. Itis likely that the permeability of the cell was al-tered by the preliminary fixation, thus allowingthe enxyme to depolymerize the DNAin the nu-clei. In order to determine further the action ofSK-SD on living leukocytes the enzyme con-centrate was added to freshly drawn human bloodin a test tube, the mixture incubated for severalhours at 37.5° C and subsequently smeared with-out fixation. Most of the nuclei in the whiteblood cells were clearly delineated by the Feulgenstain. It was apparent from the morphologicalappearance of the few cells that did not stain withthe Feulgen technique but stained readily with thecounterstain, that they were ruptured in the proc-ess of smearing. When sodium citrate was addedafter incubation and just before smearing, all thecells stained without difficulty. This was proba-bly a result of the binding of magnesium ion, anessential cofactor of desoxyribonuclease, in the

1381

ALAN J. JOHNSON

form of a poorly ionized citrate complex. Bytrial and error, it was found that "protection" ofthe smear with citrate was not necessary withtherapeutic concentrations of streptodornase.

Since SD did not act upon the nuclei of freshliving cells but caused complete depolymerizationof the DNAin fixed dead cells, its effect on exu-dates was studied where many stages of cellmaturation and degeneration have been shown toco-exist.

The microscopic examination of Feulgen-stained smearsof exudates (without fixation) disclosed two general mor-phological cell-nuclear states: (a) those whose nuclei ap-peared clear cut and sharply delineated with full nu-clear staining and (b) those degenerating cells in whichthe cytoplasm appeared intact but karyorrhexis and/orkaryolysis were taking place. The number of degenerat-ing cells divided by the total number of cells counted,times 100, was defined as the "per cent degeneration." 9

The per cent degeneration is reproducible to 1-2%:(a) when a suitable sampling technique is employed, (b)when slides are examined by the same microscopist, (c)when 300-500 cells are counted, and (d) when a mono-chromatic light source is utilized with Wratten gelatinlight filters G-15 and B-58.

The per cent degeneration was calculated from thosecells that were visible on the Feulgen stain. Since de-polymerized DNA is not stained by the Feulgen method,"degenerated" cells that had undergone extensive de-polymerization were lost to visibility by this method, al-though the "ghost forms" were visible under direct mi-croscopy, and therefore included in the total cell countsdone by the usual methods. For this reason, the sumof the per cent degeneration and the per cent of cellsstaining supravitally did not equal 100% unless the cellpopulation was moderately fresh (24-48 hours).

When SK-SD was added to purulent exudatesin zAtro the enzyme-treated specimen exhibited alow per cent degeneration of WBC's as contrasted

9 The nuclei in the degenerating cells are undoubtedlyundergoing depolymerization but by definition have al-ready undergone further depolymerization than thatwhich Pollister measured with methyl green stain (22).

TABLE I

Effect of SD on the per cent degeneration of cell nuclei inpurulent pleural exudates in vitro

Time in Per cent degeneration of Per cent degenerationhours control after incubation after incubation with SDwith heat-inactivated SD

0 52 215 76 635 97 660 99 0

with a high per cent degeneration in the controls.The extent of degeneration after enzyme exposurewas invariably reduced to 10% or less of the total,whereas the initial per cent degeneration may havebeen 90% or more.

The data in Table I were obtained on sterile empyemafluid, incubated under sterile precautions at 37.5° C.At varying time intervals 5 ml. aliquots were removedand incubated with an excess of SK-SD in a phagocyterotor for two hours at 37.5° C. Sodium citrate wasadded to both control and enzyme-treated specimens af-ter incubation, smears made and Feulgen stained, andthe per cent degeneration determined on each.

The striking decrease in the per cent of degen-erating cells after incuhation of the exudate withSK-SD was closely correlated with a comparabledecrease in the DNA of the cellular sediment(Figure 3). Experiments illustrating this wereset up as follows:

White blood cells were isolated from the peripheralblood of seven patients by a modification of the methodof Osborne (23) and a sterile exudate was utilized froma patient with tuberculous effusion. No extracellulardesoxyribose nucleoprotein was demonstrable in any ofthe samples by Feulgen staining. Consequently, anychemical evidence of DNAdepolymerization (acid solubleDNA) was referable to the degenerated nuclei. Thewhite cell preparation was incubated under sterile con-ditions at 37.5° C, aliquots subjected to an excess ofSK-SD in a phagocyte rotor for two hours, supravitalstains made, citrate added, smears made and Feulgenstained, and the per cent degeneration determined as inthe previous experiment. The cellular sediments obtainedby centrifugation were washed with citrated physiologicalsaline and the DNA content determined by chemicalanalysis. The per cent decrease in DNAwas calculatedfrom the DNAin the sediment of the enzyme-treated andcontrol aliquots.

The close correlation of cytochemical and chemi-cal methods emphasizes the value of observingchanges in the per cent of degenerating leukocytesby utilizing the Feulgen stain as a measure of SDactivity. It may also be noted in Figure 3 thatthere is no evidence of enzymatic depolymeriza-tion of the DNAin the fresh non-degenerated cellby SK-SD. This finding is supported by evi-dence presented above that staining of the viablecell nucleus by the Feulgen reaction is unaffectedby SK-SD in high titers.

Studies in which SK-SD was incubated invitro with living leukocytes obtained from theperipheral blood and exudates have also failed toreveal any effect of the enzymes on the supravital

1382

CYTOLOGICAL STUDIES WITH LOCAL INJECTIONS OF SK-SD

////0

60-o #/

40 -/

20 -

P

I/ 1

- 0 20. 40 60 s0DECREASEIN PER CENT DEGENERATION

staining and motility of the leukocytes, as well as

on their ability to phagocyte the R pneumococci.A decrease in degenerating cells has been re-

peatedly observed in vivo (Figure 4) as well as

in vitro following administration of SK-SD.The degree of change in per cent degeneration

in vitro and in vivo, respectively, is primarily a

function of the initial level, i.e., the amount ofavailable substrate (2); the higher the initial per

cent degeneration, the greater the decrement fol-lowing injection of SK-SD. For example, speci-

l00

FIG. 3. RELATION OF DECREASE IN DNA AND PERCENT DEGENERATIONOF CELL NUCLEI IN WHiTE BLOODCELLS AFTER TREATMENTWITH SK-SD

The square represents five determinations.

701

.60

E EMPYEMAS MAXILLARY SINUSITIS

HEMOTHORAXU ULCER (ED SORE)

\ ~ ~~~~~BURN(INFECTED)EF EFFUSION (TUBERCULOUS)

I\\\---10 EA ----

BHEF'sotu

Et

INJ ECTION1 3 6

TIME, HOURS

FIG. 4. EFFECT OF SK-SD ON THE PEERATION OF CELL NUCLEI IN LEUKOCYTEIN MAN

Representative data are shown fromexudates.

cm0z4

z

w,o

O2

0

4i

zW

0 *f-i

01

0

0

I

z

0

W

0

4n

INJECTION TIME, HOURS

FIG. 5a. INJECTION OF SK-SD IN A PATIENT WITH

THICK PURULENTEmPYEMASerial sampling was performed by thoraCentesis.

mens of exudates derived from areas of chronic,stagnant suppuration that drained with difficulty,such as empyema or sinusitis, exhibited a highper cent of degeneration before therapy. On the

_Na-S other hand, the free flowing material obtainedH-EFI4 from purulent lesions that had continuous open

drainage (bed sore, ulcer, infected burn) andfrom closed non-purulent lesions (hemothorax

R CENT DEGEN- and tuberculous effusion) showed a relatively lowinitial per cent degeneration. However, regard-

a variety of less of the conditions of the diseased sites, or theinitial level of the per cent degeneration, the per

1383

I.-

-Z0a:

IL

a

90

80

!ia:

z

0

IU-z

a:k!

50

40

I 30

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rI NFW I

ALAN J. JOHNSON

cent of degenerating cells in the exudate 24 hoursafter enzymatic treatment was regularly reducedto 5-10%. This phenomenon has been observedin 65 purulent and non-purulent exudates from33 patients with widely disparate diagnoses, andwas indicative of the significance of SK-SD ineliminating degenerating cell forms.

It should be clear that the changes noted abovewere acute changes, the result of an immediate re-sponse to a short stimulus. When the exudativereaction had spent itself, the new cells, too, ulti-mately degenerated.

6) Correlation of enzymatic depolymerization asdetermined biochemically wvith cellular changesin pleural exudates of patients

In order to compare the effect of SD on theextracellular desoxyribose nucleoprotein with thecellular changes that occur concomitantly, Figure5a and Figure 5b were prepared from simulta-neous chemical and cellular data derived from twopatients with thick, purulent empyema.

0 O1* OM 10.03w 0

w C

0

0

0CD

z

Immediately after the injection of SK-SD,there was a sharp fall in the extracellular Feulgen-positive material and the per cent degeneration,with a marked rise in the acid soluble desoxy-ribose nucleic acid, organic acid soluble phos-phorus, and acid soluble nitrogen liberated intothe supernatant of the exudate (Figures 5a and b).

The cytological changes occurred early andprogressed to a maximum at about 24 hours, incontrast to the faster moving biochemical response.Thus, there was a moderate increase in total whiteblood cells, and a marked increase in motile, viablecells during and immediately following the periodof greatest enzymatic depolymerization. The dis-proportionate increase in supravitally stained,motile cells may be visualized as closing the gapbetween an initial heterogeneous, non-viable,pathological cell population and the relativelyhomogeneous, viable cell population elicited by theacute enzyme action. Since this is accompaniedby an absolute decrease in the number of cellsthat do not stain supravitally immediately follow-

INJECTIONIOFIG. 5b. INJECTION OF SK-SD IN A PATIENT WITH THICK PURULENT

EMPYEMASerial sampling was performed by thoracentesis.

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CYTOLOGICAL STUDIES WITH LOCAL INJECTIONS OF SK-SD

ing the enzyme injection, it may be concludedthat the marked increase in fresh, viable cells ca-pable of phagocytosis was accompanied by a con-comitant fall in the actual number of degeneratedcells. It should be emphasized that the rapidchanges just described did not occur in a largenumber of control thoracenteses. One interestingdisparity has been observed in the cell counts ofin vitro and in vivo experiments. Following incu-bation of exudates with SK-SD in vitro, the totalcell numbers showed only a slight decrease whenthe counts were done by the usual methods eventhough Feulgen stained preparations showed amarked decrease in the per cent of degeneratingcells; and as the degenerating cells decreased, dueto enzyme action, the ghost forms increased. Inthe in sivo observations, on the other hand, theghost forms or degenerated cells were markedlydecreased. Although the phagocytosis of ghostcells or cell fragments by living leukocytes has notbeen directly observed, it seems likely that phago-cytosis, proteolysis by leukoproteinases (24, 25,26) and absorption (27) account for the oblitera-tion of the cellular forms in vivo, and with respectto the effect of enzymatic debridement accounts forthe local cleaning of tissues.

The patient's improvement following this totalresponse may be noted by the abrupt fall in totalcell count terminating the acute phase (Figure 5b)with subsequent convalescence of the area. Inthis instance also, phagocytosis by polymorphonu-clear leukocytes and possibly macrophages wasprobably of importance in the progressive diminu-tion of the total cell numbers. Simultaneous chem-ical and cytological studies of the acute phase havebeen repeatedly made with comparable results andthe subsequent course of the disease in a patientwith a bacteriologically controlled empyema usu-ally followed the pattern outlined above.

DISCUSSION

From the findings recorded in this report itis apparent that a number of changes in the localcytological picture at the site of inflammatoryexudative areas in patients, are rapidly evoked bythe introduction of SK-SD. Some of these altera-tions are manifested by the appearance in the freefluid at the site of the disease, of a considerablepercentage increase of living, active, motile leuko-cytes. From the point of view, therefore, of

cleaning the tissues at the site of disease, the ef-fectiveness of the enzymes, acting as liquefyingagents -on their specific substrates, is supplementedby the appearance of cellular elements that prob-ably contribute to debridement by phagocytosis.

It is of interest to note that a very considerableproportion of the total cell count of chronic in-flammatory exudates may be composed of degener-ating and degenerated cells. The usual methodsfor doing white blood counts give little indicationof this fact. By a combination of white bloodcounts, supravital staining, and Feulgen staining,the differentiation of living, degenerating, and de-generated cells can be made.

Inasmuch as the supravital staining, motility,phagocytosis, and the degree of polymerization ofthe DNA in the nucleus of fresh viable cells donot seem to be affected by SK-SD, the cytologicalstudies have failed to yield any evidence that theactive enzyme systems affect living leukocytes.Either the surface of the viable cell is impermeableto SK-SD or, if streptodornase is absorbed theliving nuclei inhibit depolymerization (28, 29).

SUMMARY

1. The introduction locally of streptococcal con-centrates containing streptokinase-streptococcaldesoxyribonuclease, into areas of inflammatoryexudation in patients, was followed promptly byan outpouring of living polymorphonuclear leuko-cytes which usually reached its maximum 24 hoursafter the injection. In four instances in whichboth enzymes, inactivated by heat, were injectedlocally in patients, only minimal local cellularreaction was observed. Since the concentratesemployed contained mixtures of streptokinaseand streptococcal desoxyribonuclease, the extentto which each individually contributed to thecellular outpouring has not been determined.

2. Changes in the local cytological patterncaused by the injection of the enzymes were char-acterized by a marked decrease in degeneratingand degenerated cellular forms, and a rapid risein supravitally staining,, motile, phagocytingleukocytes.

3. Depolymerization of desoxyribonucleic acideffected by treatment with streptococcal desoxy-ribonuclease caused a breaking up of clumpedleukocytes, the conglomeration of which was ap-

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ALAN J. JOHNSON

parently due to a matrix of desoxyribose nucleo-protein.

4. Liquefaction of highly viscous purulent fluidby streptococcal desoxyribonuclease, as deter-mined by preparations stained by the Feulgenmethod, resulted in the disappearance of extracel-lular desoxyribonucleic acid and the nuclei of de-generating cells. The nuclei, as well as other evi-dences of viability of living cells, remained unaf-fected. Biochemical evidence of depolymerizationaccompanied the cytological changes.

ACKNOWLEDGMENT

The author wishes to acknowledge the capable techni-cal assistance of Miss Doris Bitz and Mrs. Vesta Knaub.

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