Virus-induced alterations of lymphoid tissues

CELLULAR

IMMUNOLOGY

333-354

1,

Virus-Induced I. Modification

(1970)

Alterations of the Recirculating by Newcastle

F. WOODRUFF*

JACK Department New

York

of Pathology,

10016,

of

New

Virus 1

JUDITH

J. WOODRUFFS

York

University School of Medicine, New York, of Medicine and Pathology, CornellUniversity New York, New York 10021

and the Departments Medical

Tissues

Pool of Small Lymphocytes

Disease AND

Lymphoid

College, Received

April

1, 1970

Intravenous inoculation of Newcastle disease virus (NDV) altered the character of the recirculating pool of small lymphocytes. In mice severe lymphocytopenia and depletion of small lymphocytes from the deep cortex of lymph nodes and from the periarteriolar lymphoid sheaths of the spleen were found 24 hr after virus challenge. By 72 hr the concentration of blood lymphocytes was normal and the areas in the lymph nodes and spleens which were depleted at the earlier interval showed an increased concentration of lymphocytes. In virus-treated rats it was also shown that depletion and repopulation of blood with lymphocytes were paralleled by similar changes in the output of small lymphocytes from the thoracic duct. Thus, a marked deficit of small lymphocytes in the thoracic duct lymph was observed when rats were cannulated at the time of virus challenge; the output of small lymphocytes in the lymph was normal when rats when cannulated 72 hr after virus inoculation. Evidence was obtained that the ability of NDV to modify the recirculating lymphocyte population was independent of its capacity for complete viral replication and independent of functioning adrenal gland tissue. Possible mechanisms that may account for these abrupt and marked shifts in the concentration of recirculating lymphocytes in the blood, lymph, and lymphoid tissues are discussed. INTRODUCTION Several viruses cause transient lymphocytopenia ( 1-S). This condition has been observed ijn both man and animals and is variably associatedwith a reduction in the concentration of other blood cells. Little is known about the mechanism(s) (4, 7) and consequence(s) of virus-induced lymphocytopenia. The nature of the lymphocytic abnormality has not been defined, and it is not know;n what property(ies) 1 This work was supported in part by National Institutes of Health Grants .5Tl-GM00127, GM00078-11, and AM-10252-14. 2 Present Address : Department of Pathology, Cornell University Medical College, New York, N.Y. 10021. 3 Present Address : Department of Microbiology and Immunology, Downstate Medical Center, Brooklyn, N.Y. 11203. 333

334

WOODRUFF

AND

WOODRUFF

of the virus is responsible for producing this state nor whether direct lymphocytevirus interaction is involved. The factors determining the deficit and reconstitution of blood lymphocytes during viral disease are of jnterest since there is evidence that these cells may participate directly in host defense against viral infection (9, lo), and since viruses can alter immune responses of the host (1 l-14). Newcastle disease virus (NDV) challenge produces marked lymphocytopenia in mice. (Woodruff, J. F., and Kilbourne, E. D., unpublished observations). The present report describes in NDV-challenged animals the rapid decrease in the size of the circulating pool of lymphocytes and the concommittant depletion of lymphocytes from those areas of lymphoid tissues through which lymphocyte traffic is known to occur (15). It appears that this effect of NDV is largely independent of adrenal cortical secretion and is not dependent on the ability of this virus to achieve a complete cycle of replication. MATERIALS

AND

METHODS

Anis&s. Specific pathogen-free (SPF) male albino mice (Manor North, Staatsburg, N.Y.) were received in the laboratory at 60 days of age and were housed in individual cages for at least 1 week before use. The mice were fed a regular diet and water ad libitum. In some experiments male, Sprague-Dawley rats weighing 200-250 g were used. Org+ weights. Organs were fixed in 10% formalin and then weighed to the nearest 0.1 mg. Histology. Cut sections of the formalin-fixed organs were stained with hematoxylin and eosin (H & E). Virus. Newcastle diseasevirus (NDV) (H’ ICk man strain) was grown in the allantoic fluid of lo-day-old embryonated eggs. Nearly all allantoic fluid virus preparations were dialyzed at pH 7.0 for 24 hr before further processing. Four types of NDV preparations were used in these experiments: (a) virus in allantoic fluid, (b) virus obtained after ultracentrifugation of allantoic fluid, (c) virus partially purified on a sucrose cushion, and (d) virus in allantoic fluid which was inactivated by ultraviolet (uv) light or heat. In preparation (b), the allantoic fluid was centrifuged at 100,OOOgfor 2-3 hr and the virus pellet then resuspended in 1X phosphate-buffered saline (PBS), pH 7.2. In virus preparation (c) , virus-infected allantoic fluid was passedthrough coarse gauze and then layered on a 60% sucrose solution and covered by a lO~o/osucrose solution. After centrifugation at 20,000 rpm for 30 min (No. SW27 rotor), the virus layer was removed. This virus, as measured by hemagglutination units, was concentrated lo-fold by this method. In preparation (d), 15-ml alicluots of virus in allantoic fluid were placed in glass Petri dishes and exposed to uv light for 5 or 15 min (7 in. from the light source; 7% W G.E. “sterilamp”). During this treatment, the Petri dishes were placed in an ice bath which effectively prevented any detectable decreasein the hemagglutination or neuraminidase titers of the virus. Heated virus was prepared by incubating 5 ml of virus in allantoic fluid in a 56” water bath for 30 miln. Virus-free allantoic fluid was prepared by passing the allantoic fluid virus suspension [containing lo8 egg infective dose,, (EID,,)/O.l ml] through a lOO-rnp

VIRUS-INDUCED

ALTERATIOXS

OF

LYMPHOID

TISSUES

335

Millipore filter. Infective virus was not recovered when this filtrate was inoculated undiluted into eggs. Y&U (seed) titrations. Each preparation of NDV was characterized by determining its hemagglutination titer (H.A.) and EID,,. The H.A. titrations were performed on serial dilutions (with PBS) of the virus which were incubated at 4” with human type 0 Rh-positive red blood cells (RBC) (16). EID,, was determined by inoculating the allantoic fluid of IO-12-day-old embryonated eggs with 0.1 ml of lo-fold dilutions of the virus in antibiotic saline solution (ABSS). Three to four eggs were used at each dilution. After a 4%hr incubation period at 37”, the allantoic fluid in each egg was examined for hemaggluti/nating activity at a 1 : 4 dilution and the EID,, calculated. The neuraminidase (NANASE) content of some virus seeds was also determined using the Warrenhoff method (17). Organ and seYu7lz virus UEYS. The titers of NDV present in the sera and organs of mice were determined in the following manner. The blood for serum virus titers was obtained by open cardiac puncture under Nembutal anesthesia. The blood was allowed to clot at 4” for several hours and the serum removed after centrifugation. The animals were then killed by cervical fracture and the spleen, liver, thymus, and axillary lymph nodes removed aseptically. These organs were suspended in ABSS, ground in glass tubes using Teflon grinders, and then centrifuged at 8000 rpm for 10 min. Serial lo-fold dilutions (in ABSS) of these specimens were subsequently inoculated into eggs (0.1 ml) as described above, and the EID,, determined. NDV antiserum. Antiserum to NDV was prepared by inoculating a 2-kg rabbit in the footpad with 0.2 ml of NDV in complete Freund’s adjuvant. The NDV had been partially purified on a sucrose cushion and had an EID,, titer of 10IO/O.l ml and a H.A. titer of l/4096 units/O.4 ml. The virus and complete Freund’s adjuvant were thoroughly mixed in equal volumes before inoculation. Approximately 40 days later, the rabbit was inoculated intravenously (iv) with 0.3 ml of the same virus preparation without Freund’s adjuvant. Ten days later, venous blood was obtained and allowed to clot at 4”. After centrifugation of the blood at 1000 rpm for 15 min, the serum was removed, heated at 56” for 30 min, and treated with periodate solution to destroy complement and nonspecific (viral) inhibitors (16). Normal rabbit serum (NRS) was processed in the same manner. Following this, the antiserum and NRS were absorbed twice with washed, packed human type 0 Rh-positive RBC. Serial 2-fold dilutions of 0.2 ml of these sera were then made in PBS and to each tube 0.2 ml of NDV containing 32 hemagglutination units was added. After incubation at room temperature for 30 min, 0.4 ml of human type 0 Rh-positive RBC were added and H.A. activity determined (16). The rabbit antiserum was then found to have a hemagglutination inhibition (H.I.) titer of l/8192/0.2 ml. Mode of inoculation. All injections, except as noted in the text, were made via the lateral tail vein. Virus-inoculated mice were injected with 0.4 ml of each virus preparation. Control mice were injected with 0.4 ml of virus-free allantoic fluid, PBS, or a 3.5% sucrose solution, as indicated in the text. Rats lightly anesthetized with ether were inoculated with O.GO.8 ml of virus suspension or PBS. Hematologic detevnzinafiom. Blood leukocyte counts and hematocrits were performed in the usual manner on blood obtained from superficially cut tail veins of unanesthetized mice and ether anesthetized rats. Blood smears were stained with

336

WOODRUFF

AND

WOODRUFF

Wright’s or Giemsa stain and differential leukocyte counts were done by counting at least 200 cells. The absolute number of lymphocytes and granulocytes per mm3 of blood was then calculated. In some severely leukocytopenic mice, however, it was not possible to enumerate more than 50-100 cells. Because of the diurnal variation in the concentration of peripheral blood lymphocytes, these determinations were carried out between 10 :00 a.m. and 2 :00 p.m. Lymphocyte counts obtained from virus-treated animals have been expressed as the percentage of lymphocyte counts of control mice (1000/o) which were simultaneously performed. Experimental design. Groups of virus-inoculated and control animals were weight-matched at the beginning of each experiment and were maintained in the same environment for the duration of the experiment. At varying intervals after injection, blood was obtained from three to six virus-treated and three to six control animals for hematologic studies. The animals were then sacrificed and the axillary lymph nodes, spleen, and thymus removed. Deter&nation of the recovery of lymphocytes in the thoracic duct lymph of virus-inoculated and uninoculated rats. The thoracic ducts of rats were cannulated by the usual method (15). After surgery. the animals were injected with either NDV or PBS via the lateral tail vein. The unanesthetized rats were maintained in Bollman cages with free accessto commercial rat cake and 5% sucrose in saline. In some experiments virus or PBS was injected intravenously 72 hr before cannulation of the lymph duct. When free flow was established, lymph was collected for 12 hr at room temperature in flasks containing 20 ml of isotonic, heparinized saline (20 units/ml). Adrenalectomy. In some experiments, mice were bilaterally adrenalectomized prior to virus challenge. The mice were anesthetized with Nembutal and the adrenals were removed as previously described (10). The mice were then inoculated subcutaneously with 1.0 ml of isotonic saline and then returned to individual cages. The animals were fed a regular diet and saline ad lib&m. All adrenalectomized animals were used in experiments 7-12 hr after surgery. Statistical analysis. A variance technique (Student t test) was used to analyze the differences in average hematocrits, blood lymphocyte and granulocyte counts, thoracic duct outputs, and body and organ weights. RESULTS

Mice inoculated intravenously with 4 X lo8 EID,, NDV exhibited only mild and transient signs of illness. By 24 hr after injection, virus-treated mice had lost approximately 10% of their body weight and in general appeared less active than control mice. By 3-6 days after inoculation no difference in the weight and activity of virus-treated and control animals was noted. Recovery of infective virus. The axillary lymph nodes, spleen. thymus, liver, and serum of mice were assayed for residual infective virus 12, 24, and 72 hr after virus inoculation (4 X lo8 EID,, NDV, iv). Virus was demonstrable in the majority of spleens (4/6) at 12 hr, but only in l/6 thymi and l/6 livers during the first 24 hr after injection. Virus was not recovered in lymph nodes and sera at the lowest dilutions tested (10-l.” and lO-l.O, respectively). Virus, when detected in organs, was present in a very low average titer (5 101.sEID,,/O.l ml). By 72 hr

VIRUS-INDUCED

ALTERATIONS

OF

LYMI’HOID

337

TISSUES

infective virus was not detected in any specimen. These results are in accord with previous reports that NDV does not achieve a complete cycle of replication in adult mice (18-20). Peripheral blood lymphocyte and granulocyte counts in mice inoculated with NDV in allantoic fluid. Lymphocytopenia was found in all mice 12, 24, and 48 hr after intravenous inoculation of allantoic fluid containing 4 X lo8 EID,, NDV (see Table 1) . In these animals, blood lymphocyte counts were approximately 305% of control values at 12 and 24 hr and 50% of control values at 48 hr after inoculation. Duripg the next 24 hr the concentration of blood lymphocytes in virus-treated mice rose markedly. Thus, at 72 hr after inoculation, the lymphocyte counts of these mice were equal to or nonsignificantly different than that found in control mice. The average granulocyte count of the virus-injected mice was significantly elevated at 12 hr but not at subsequent intervals. These results are also shown in Table 1. The variation in the mean lymphocyte and granulocyte counts observed among the control (PBS-injected) mice on different days was not statistically significant. Effect

of different

preparations

of NDV

on peripheral

blood

lymphocyte

counts.

In subsequent experiments, preparations of NDV were used which had been subjected to different degreesof purification. For some experiments virus was concentrated by ultracentrifugation and resuspended in PBS ; in others, virus was partially purified on a 60% sucrose cushion. Comparison of the lymphocytopenic effect of the various NDV preparations at a constant dose of infective virus is shown in Fig. 1. All of these viral preparations produced approximately the same degree of lymphocytopenia. In contrast, no difference was found between blood lymphocyte values obtained in the PBS-injected mice and in mice inoculated with virus-free allantoic fluid. These results are consistent with the view that lymphocytopenia is a consequenceof the activity of the virus and not some other component of allantoic fluid. Characteristics and specificity of blood lymplzocyte response to NDV. The degree of lymphocytopenia was found to be directly related to the number of infective virus particles injected. Thus, when mice were inoculated with varying concentrations of virus ranging from log to 10-l EID,, NDV/O.l ml, significant lymphocytopenia was only noted in those animals challenged with 4 X log, 4 X lOa, or 4 X lo7 EID,, NDV (see Fig. 2). Also the degree of lymphocytopenia was greater in mice inoculated with 4 X lo9 than with 4 X lo1 EID,, NDV (p < .OS). The effect of NDV on lymphocytes was also influenced by the route of inoculation. As shown in Table 2, NDV injected intravenously or intraperitoneally induced lymphocytopenia while no significant change followed subcutaneous challenge. It was also found that NDV-treated mice challenged 14 days later with a second dose of virus (1 X 10’ EID,, NDV inoculated each time) developed lymphocytopenia which was not significantly different from that observed in recipients not previously exposed to this virus. Since the fate of NDV in mice was altered by the incubation of the virus with specific rabbit antisera before intravenous inoculation (21), it was of interest to determine the effect of this treatment on NDV-induced lymphocytopenia. As shown

2,984

2,712 4,277 1,962

14,583

16,938 13,323 13,488

Control

(’

<.02

< .Ol

12

16,760 562

18,355 9,600 24,854 14,230

4,135 28

3,920 2,400 6,647 3,571

Virus

6

3,925 2,240 2,435 1,495 1,846 2,920 3,582 3,472 1,505 2,602

13,675 15,260 23,465 1,455 15,404 25,267 13,893 15,978 17,070 16,385

Cot1tr01

NS

< .Ol

24

a Control mice (PBS-inoculated). ‘JVirus-inoculated mice (4 X lo8 EIDao NDV in allantoic fluid). c P, probability of difference between mean cell counts of virus-treated d NS, nonsigniticant difference.

Mean 70 of control P

Granulocytes ( N/mms)

PC

Mean To of control

Lymphocytes (N/mm3)

I

after

and control

12,347 3,626 223 1,678 3,141 852 1,963 2,892 1,942 3,185 122

4,028 7,849 1,702 2) 347 5,234 5,148 2,612 6,433 4,108 4,385 27

Virus

Hours

2,540

____

being

2,680 2,306 1,550 2,120 3,434 3.151

19,027

14,549 19,470 24,244 23,450 13,581 18,866

Control

animals

-~-

inoculation 48

1,908 75

1,432 1,411 1,203 3,672 2,684 1,046

9,067 48

8,379 9,018 9,039 9,447 9,878 8,641

Virus

__-

INOCULATED WITH

significant.

NS

< .Ol

PEKIPHEK~~L R~oon LYMPHOCYTE AND GRANULOCYTE COUNTS IN MICE NEWCASTLE DISEASE VIRUS IN ALLANTOIC FLUID

TABLE

2,510

2,811 3,075 2,048 1,738 2,597 2,792

12,974

12,789 12) 030 14,073 14,416 9,428 15,108

Corltrol

NS

NS d

72

3,515 140

3,048 3,494 4,239 4,285 1,673 1,394 6,470

14,985 11s

12,230

9,331

8,420 16,969 32,097 15,821 10,027

Virus

__-

z =:

2 8

VIRUS-INDUCED

ALTERATIONS

OF LYMPHOID

339

TISSUES

125 t

I I

01 DAYS

I

2 AFTER

I

3

INOCULATION

1. Blood lymphocyte counts of mice inoculated with different preparations of NDV. Lymphocyte counts obtained in control mice (inoculated with PBS) are expressed as 100%. o-o, Recipients of KDV-infected allantoic fluid (4 X 10s EID,,). Each point represents the mean value listed in Table 1. O-O, Each recipient injected with 4 X 10s BID,, of virus that was ultracentrifuged and resuspended in PBS. Each point represents values obtained in 6-10 uninjected and &IO virus-injected mice. x-x, Each recipient injected with 4 X 10s RID,, of virus that was partially purified on a sucrose cushion. Control injected with 3.5% sucrose solution. Each point represents mean value in five control and five virus-inoculated mice. A-A, Each recipient injected with m-irradiated NDV containing residual infectivity of 101 EID,,/O.l cc. Each point represents mean of four control and five virus-inoculated mice. n---c], Recipients of virus-free allantoic fluid; each point represents mean of five experimental and five control mice. FIG.

in Table 3, the degree of lymphocytopenia was significantly less when NDV in allantoic fluid was incubated in vitro with rabbit antiNDV antisera prior to injection. Incubation of NDV with NRS did not reduce its lymphocytopenic effect. Also injection of antisera or NRS alone did not significantly alter the mean lymphocyte counts as compared to values obtained in PBS-treated mice. In order to obtain further information about the nature of the lymphocyte abnormality induced by NDV challenge, mice were studied at intervals of 6 hr to 21 days after inoculation of virus (4 X lOa EID,, NDV, prepared by ultracentrifugation). The rate of induction of lymphocytopenia as shown in Fig. 3 was rapid ; by 6 hr after virus challenge significant lymphocytopenia was present. Lymphocytosis was observed at 6 and 10 days, but at later intervals the lymphocyte counts of virus-inoculated and control mice were approximately the same. No difference was detected in the morphology of blood lymphocytes in NDVtreated and control mice. In both groups of animals nearly all lymphocytes were small and medium-sized cells. Recovery from the lymphocytopenic effect of NDV was not associated with the appearance of atypical lymphocytes in the peripheral blood.

340

WOODRUFF

IO3 P*

co1

IO8

AND

IO7

c.01

co1

DOSE

OF NW

WOODRUFF

IO0

Id

N.S.

N.S.

INOCULATED

IO’ N.S.

Id2 N.S.

PBS (conlrol)

(EID,,)

FIG. 2. Blood lymphocyte counts 12 hr after inoculation of mice with 0.4 ml of varying doses of infective NDV. NDV was concentrated by ultracentrifugation and resuspension in PBS. The designated virus doses represent EID,,/O.l ml. Each bar represents the mean cell count found in three mice. *Probability of difference between average lymphocyte counts in virus-treated and control mice being significant. N.S., nonsignificant difference.

There was no evidence that NDV caused anemia or granulocytopenia 3). Significant granulocytosis was found only at 6 and 12 hr after virus The hematocrits of NDV-treated mice were significantly elevated at 24 but were nonsignificantly different from control values at later intervals. vated hematocrits and the abrupt loss in weight described above suggest TABLE EFFECT ON ABILITY Inoculum route Blood lymphocyte (Nlmm3)

Mean y& of control fid

c

b

2

01; DIFFER&NT ROUTES OF INOCULATION OF NDV TO INDUCE LYMPHOCYTOPENIA PBS, iv

counts

(see Fig. injection. and 48 hr The elethat these

12,636 9,011 13,818 7,334 10,699

NDV,a iv

NDV, ip

384 4,643 4,129 3,505 3,165 29.6 <.Ol

a Each virus-inoculated animal received 4 X lo* EIDjO cushion. * Blood lymphocyte counts obtained 24 hr after inoculation c Percentage of values obtained in PBS-injected animals. d Probability of difference in average lymphocyte counts mice being significant.

ND\‘, SC

2,388 3,625 3,099 3,089 3,050 28.5 <.Ol NDV

between

partially

13,000 5,093 11,232 5,700 8,756 81.8 NS purified

virus-treated

on sucrose

and

control

VIRUS-INDUCED

ALTERATIONS

OF LYMPHOID

TABLE INFLUENCE OF A4x~~ THE LYMPHOCYTOPENC NDV + PBS, 6 animals Blood

lymphocyte

count

2,293 1,876-2,647

PIC PBS, 7 animals count

3 NDV

ah~~~~~~~~~ ON EFFECT OF NDV NDV + NRS,a 5 animals

NDV

+ Antiserunr,n 9 animals

*

W/mm? Mean Range

Blood lymphocyte (N/mm3) 1lean Range p2e p;f

341

TISSUES

3,510 1,254-5,023 NS NRS,” 5 animals

6,536 4,282-9,450 <.Ol Antiserum, 8 animals

d

b 9,317 7,114-12,702 <.Ol

12,872 8,456-16,851 < .Ol NS

10,063 6,289-16,101 .02 > p > NS

.Ol

a 2.5 cc of NDV in allantoic fluid (1O8.3 EIDr,o/O.l cc) incubated in vitro with either 0.3 cc NDV antiserum, 0.3 cc NRS or 0.3 cc PBS. After 30 min at room tempsrature each recipient transfused iv with 0.4 cc of the indicated inoculum. * Values obtained 24 hr after inoculation. c pi Comparison of values obtained in recipients of NDV + PBS to that found in recipients of NDV + NRS and of NDV + antiserum. d 0.3 cc antiserum or NRS incubated at room temperature with 2.5 cc PBS. After 30 min 0.4 cc of the indicated inoculum injected into each animal. e p:, comparison of values obtained in NDV + PBS vs. PBS, ND\’ + NRS vs. NRS and NDV + antiserum vs. antiserum. f pa, Comparison of values obtained in recipients of NRS and of antiserum vs. PBS.

mice were dehydrated during the early period following virus inoculation. The concentration of large mononuclear cells in the blood of virus-treated and control mice was not significantly different at any interval tested. Ability of w&radiated and heated NDV to induce lyynphocytopeniu. The properties of NDV which were responsible for the development of lymphocytopenia are not known. Studies were carried out to determine if this effect was related to the ability of NDV to replicate normally. This characteristic of NDV was measured by egg infectivity. Aliquots of NDV in allantoic fluid with an infectivity titer of 108,y EID,,/O.l ml were exposed to uv irradiation for 5 min; the resulting preparation had an infectivity titer of < 1Ol.O EID,,/O.l ml. This preparation of NDV induced lymphocytopenia to approximately the same extent as nonirradiated NDV (see Fig. 1 and Table 4). In other experiments NDV in allantoic fluid ( 10s.3 EID,,/O.l ml) was uv irradiated for 15 min and the resulting preparation had no detectable infectivity (as determined by assay of five separate aliquots) . Inoculation of mice with this virus preparation, as shown in Table 4, also induced marked lymphocytopenia which was nonsignificantly different from that observed in mice after inoculation of NDV with an infectivity of 4 X 10s.3 EID,,. Although uv irradiation destroyed the ability of NDV to achieve a complete cycle of replication in ovo, there

342

WOODREFE

r: 2 ; z .l

AND

WOODRUFF

lWjj

I

500

BLOOD GRllNULOCYTES

400

H&

k D9YSlb”TIME

AFTER

I3 ‘“‘2,

INOCULATION

FIG. 3. Differential effect of intravenously inoculated NDV on peripheral blood lymphocyte and granulocyte counts and on hematocrits. Mice inoculated with 4 X 108 EID,, NDV. At each interval 6-12 control (inoculated with PBS) and 6-12 virus-injected animals were examined.

was no evidence that the hemaggIutinating and neuraminidase activities of NDV were altered by this treatment. While the lynlphocytopenic effect of NDV was preserved after uv irradiation, the same virus preparation did not induce the granulocytosis which was observed after inoculation of fully infectious virus. Since the ability of NDV to replicate in eggs was not required for the production of lymphocytopenia in mice, efforts were made to determine the roles of the viral hemagglutinating and neuraminidase proteins in the production of this lesion. Purified NDV was iincubated with trypsin since this enzyme has been reported to release neuraminidase from the surface of certain strains of NDV (22). However, the neuraminidase activity of the strain of NDV used in the present experiment was not susceptible to this treatment. Therefore, NDV in allanoic fluid was incubated at 56” for 30 min. Following this treatment, the hemagglutination, neuraminidase , and egg infectivity titers of NDV were markedly depressed. This heated virus preparation, as shown in Table 4, did not produce lympho~ytope~lia.

108 3 Neg. d

108.3 103.7

PBS NDV uv 15 mill

PBS NDV heated

NDV,

NDV,

cc

HA

256 <4

512 512

512

units/O.4

of virus

NDV

COUNTS

cc

NANASE

preparations

IdYhl~HOCYm UV-TREATED

,360 ,041

,390 ,360

activity

a

3 3 4

7 4 7

4 5

No. of animals

OF INFECTIOCS

groups

being

significant.

14,816 4,060 10,112

16,370 3,982 6,274

12,240 4,574

Mean blood lymphocyte count (iv/mtn3)

assayed.

24 HK AFTER INOCULATION OR HEATED NDV

4

n Neuraminida~e activity. values are the O.D. readings at a wavelength of 549 p for 0.1 cc of virus b Percentage of mean blood lymphocyte count of PBS-inoculated mice. c p, probability of difference between mean lymphocyte counts in the virus-treated and in control 1 neg, No infectivity for eggs using undiluted virus suspension e 56” for 30 min. i NS, nonsignificant difference.

e

lo’.0

EID,i,/O.l

BI,cI~~

Characterization

NDV,

-__~~

OF >~EAN

PBS uv 5 min

InoculutII

~o~IPARISON

TABLE NDV,

NS=f

<.Ol

100 27 68

< .Ol < .Ol

< .Ol

PC

100 27 38

100 37

% of control values *

2 ki 2 c% s m

2 z

$ 2 $ 2 0 z tn 9“I

Q

3 c

5 A

2

NS

< .Ol

d

NDV). cell count

Died 1,768 7,544 1,892 2,820 2,806 53

2,482 3,130 1,833 3,956 2,850 30

Died

v ‘,

1

GRANULOCYTE

a Control mice (PBS-inoculated). b Virus-inoculated mice (4 X lo* EIDw c p, probability of difference in average d NS, nonsignificant difference.

P

Mean y0 of control

Granulocytes iN/mm3)

PC

4,531 5,632 4,752 3,053 8,575 5,309

6,968 9,023 10,025 9,723 9,367

Mean 70 of control

11,094

(N/mm3)

Ca

AND

Experiment

LYMPHOCYTE

Lymphocytes

BLOOD

s, 939

C

NS

< .05

virus-treated

1,652

1,288

1,069 2,598

12,400

5

\

-

-

C

animals

inoculation

being

.~

Experiment

ADKEXALECTOMIZED

and control

3,505 212

5,270 2,783 2,536 3,432

32

3,960

4,401 2,619 4,446

4,374

2

Hr after

IN BILATEKALLY

Experiment

18,560 9,701

between

12

COUNIY

TABLE

v

significant.

Died Died Died

24

CHALLENGED

Died Died

Died Died Died Died

Died

1

MICE

2,920

1,350 3,552 2,860

15,025

15,023 20,982

9,069

C

ND\’

<

05

NS

Experiment

WITH

v

2,607 83

Died 2,723 3,534 1,563

21

3,104

1,528 2,095 5,688

Died

2

< 8 ii c: 7

E s : F3

2

F

VIRUS-INDUCED

ALTERATIONS

OF LYMPHOID

TISSUES

345

Effect of bilateral adrenalectomy on the ability of infectious and w-treated NDV to induce lymphocytopenia. Lymphocytopenia is found after injection of ACTH or adrenal cortical steroids (23), and it has been suggested that viral-induced lymphocytopenia may be mediated by stimulation of the adrenal glands (4). In order to determine if the effect of NDV on lymphocytes required intact adrenal glands, bilaterally adrenalectomized mice were injected with either 4 X lo8 EID,, NDV (ultracentrifuged and resuspended in PBS) or with PBS. As shown in Table 5, NDJ7-injected, adrenalectomized mice were severely lymphocytopenic 12 and 24 hr after challenge; at these intervals the virus-treated mice had blood lymphocyte counts which were 32 and 21%, respectively, of control values. Thus, the degree of lymphocytopenia was approximately the same in adrenalectomized and in intact mice at a constant viral dose. While spontaneous deaths in virus-infected, intact mice were extremely rare. nearly all adrenalectomized mice inoculated with NDV died 24 to 48 hr after inoculation. This high incidence of death, but not the lymphocytopenia, was prevented by inoculating adrenalectomized mice with uv-irradiated NDV (residual infectivity 10” EID,,/O.l ml). At 24 hr, such mice had blood lymphocyte counts which were 25% of values obtained in adrenalectomized mice injected with PBS. Although bilateral adreinalectomy did not alter the ability of NDV to induce lymphocytopenia, it did prevent the lymphocytopenic effect of ACTH indicating that these animals did not retain functioning adrenal tissue. Thus, 10 hr after subcutaneous injection, the blood lymphocyte concentrations of intact mice receiving 10 units of ACTH (Purified Cortrophin Gel, Organon, Inc.) or PBS were 3321/mm” (mean of five mice) and 13,440/ mm3 (mean of five), respectively. Adrenalectomized mice injected with ACTH (six animals) or PBS (seven animals) had mean blood lymphocyte counts of 13,556 and 11,918/mm3, respectively. Changes in lymphoid organs following virus inoculation. The average weights of the spleen, thymus, and lymph nodes were nonsignificantly different in NDVtreated and colntrol mice from day 1 to day 6, except at day 6 when the spleen weights of virus-treated animals were significantly increased. Characteristic lesions were found in the majority of axillary lymph nodes and in all spleens 24 hr after inoculation of mice with 4 X lo* EID,,, SDV. A marked to moderate depletion of small lymphocytes from the deep cortex of 68% of the 22 axillary lymph nodes examined was found (See Figs. 4 and 5). Numerous empty spaces were often present in the deep cortex and these spaces were outlined by an intact reticulum network. Cellular debris was infrequently detected in this region. A well defined rim of normal superficial cortex, containing nodules, was present in these lymph nodes. The spleen of every animal inoculated with virus (lO/lO) showed a moderate to marked decrease in the concentration of small lymphocytes in the areas of white pulp adjacent to the central arterioles (See Figs. 6 and 7). Collections of cellular debris were seen in areas of lymphocyte depletion. These foci of lymphocyte depletion were separate and distinct from the germinal ceinters. The splenic sinusoids were engorged with erythrocytes. In contrast to the above findings in lymph nodes and spleens, histologic changes were infrequently found in the thymi of these virus-inoculated mice. Lesions were seen in only two of seven thymi examined and consisted of several punctate areas

346

FI’ G. 4. Axillary FING. 5. Axillary show ring depletion E. X32. H&

WOODRUFF

lymph

node of control

AND

WOODRUFF

(PBS-inoculated)

mouse.

H. & E. X 45.

NDV lymph node 24 hr after intravenous inoculation of 4 X lo8 EID;, of lymphocytes from the deep cortex. The superficial tortes is ncwmal.

VIRUS-INDUCED

ALTERATIONS

OF

LYMPHOID

FIG. 6. Lymphoid follicle of spleen of control (PBS-inoculated) Note aggregation of lymphocytes around central arteriole.

TISSUES

347

mouse. H & E. X435.

FIG. 7. Spleen 24 lx after inoculation of 4 X 10s EID,, NDV. The periarteriolar depleted of lymphocytes. The germinal center is intact. H & E. X 100.

sheath is

348

WOODRUFF

AND

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in the cortex containing large, pale cells, pyknotic nuclei, and cellular debris. These alterations are similar to but less extensive than those described following intracerebral inoculation of NDV (24). The histologic appearance of the spleens and lymph nodes at 72 hr after inoculation of virus was markedly different from that observed at 24 hr. At the later interval the character of the splenic white pulp was essentially normal in all virustreated (6/6) mice. In particular, the previously observed lymphocyte-depleted areas in the periarteriolar regions of the spleens were now filled with cells that histologically resembled small lymphocytes. At 72 hr some lymph nodes from virustreated animals did show a slight deep cortical depletion of small lymphocytes. However, the concentration of lymphocytes in this region in most of the lymph nodes appeared greater than that observed in recipients sacrificed 24 hr after injection of virus. All the thymi examined at 72 hr after inoculation were normal. Inoculation of mice with uv-irradiated NDV (no residual infectivity for eggs) also caused, at 24 hr, depletion of lymphocytes from the deep cortex of lymph nodes and from the periarteriolar regions of the spleen. Bilaterally adrenalectomized mice inoculated with infectious virus (4 X lo* EID,,) showed, at 24 hr, marked depletion of lymphocytes from the periarteriolar regions of the spleen (Fig. 8). The extent of lymphocyte depletion in these spleens was often more severe than that found in intact, virus-treated animals. In the virus-inoculated adrenalectomized mice, there was an obvious decrease in the amount of cellular debris found in the white pulp of the spleen as compared to that observed in intact, virus-treated animals. The changes in the deep cortex of the lymph nodes were often less severe than that shown in the virus-inoculated intact animals. The output of lymphocytes in the thoracic duct lymph of control and virustreated rats. Studies were carried out to determine if, after NDV challenge, changes in the cellular content of thoracic duct lymph correlated with virus-induced alterations in the concentration of lymphocytes in the blood and lymphoid tissues. These experiments were performed in rats since mice tolerated poorly the combined treatment of thoracic duct drainage and virus challenge. It was found that changes in the blood lymphocyte concentration of rats intravenously inoculated with 10”.4-1010EID,, NDV were comparable to that observed in mice challenged with 4 X lo8 (i.e., 108.6) EID,, NDV. Thus, virus-treated rats showed, at 24 hr, a 60 - 70% reduction in the concentration of small lymphocytes in the peripheral blood while at 72 hr the blood concentration of these cells was the same as that observed in control (PBS-injected) rats. When rats were inoculated with NDV at the time of cannulation, there was a marked reduction in the number of small lymphocytes recovered in the first 12-hr lymph collection (Table 6). The mean hourly output of lymphocytes from virustreated rats was 23.8 X 106and from PBS-injected rats, 59.4 X 10G.Although virus-injected rats showed an increased number of erythrocytes in the lymph, there was no evidence of clots in the cannulae and the flow of lymph in all animals was brisk. In contrast to the severe deficit of lymphocytes in the lymph induced by NDV under these conditions, the output of lymphocytes was normal when rats were cannulated 72 hr after virus challenge; in these animals the mean output of lymphocytes in the first 12 hr lymph collection was 63.0 X 106/hr.

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349

TISSUES

FIG. 8. Spleen from a bilaterally adrenalectomized mouse, 24 hr following the of 4 X 10s EID,, NDV. There is a marked depletion of cells from the periarteriolar sheath. Cellular debris is not a prominent finding. H & E. X 225.

inoculation lymphoid

At both time periods following inoculation, 89-94s of the cells collected from the thoracic duct of virus-treated and control rats were small lymphocytes and more than 98% of these cells excluded trypan blue. The deficit of lymphocytes in the blood and lymph of virus-inoculated rats was

TABLE THE

EFFIXX OF NDy ~~oc~L.4~10~ ON THE OF I
Thoracic Inoculum

PBS Virus Virus

d e

No. of animals 12 5 5

6

Lymph 107 88 120

duct

\-olume (75-160) (65-126) (98-140)

output No.

c

OLTKT

7 13 296 7.56

a ~--~ of cells X lo6 (590-966) (158-359) (480-l 110)

a Values are for the first 12-hr collection. * Probability of difference between mean cell count of PBS-inoculated being significant. c Range of values obtained. d 109.55-1010 EID50 NDV (ultracentrifuged and resuspended in PBS) at the time of thoracic duct cannulation. e lOlo EIDjo NDV intravenously inoculated 72 hr before cannulation

y. Small lymphocytes

Pb

89-93 90-92 90-94

<.Ol NS

and virus-inoculated

rats

intravenously

inoculated

of the thoracic

duct.

350

WOODRUFF

AND

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also related to the dose of virus injected; thus, the blood lymphocyte concentration and the output of small lymphocytes in the thoracic duct lymph were normal 24 hr after inoculation of rats with 5 X 10s EIDSo NDV. DISCUSSION The results of the current experiments demonstrate that Newcastle disease virus (NDV), intravenously inoculated, altered the character of the recirculating pool of small lymphocytes (15, 25, 26). In mice, severe lymphocytopenia and depletion of small lymphocytes from the deep cortex of lymph nodes and from the periarteriolar lymphoid sheaths of the spleen were found 24 hr after virus challenge. By 72 hr the concentration of blood lymphocytes was normal and was associated with an increase in the lymphocyte population in the areas in the lymph nodes and spleens which were depleted at the earlier interval. In virus-treated rats it was also shown that depletion and repopulation of blood with lymphocytes paralleled similar changes in the output of small lymphocytes from the thoracic duct. Thus, a marked deficit of small lymphocytes in thoracic duct lymph was observed when rats were cannulated at the time of virus challenge ; the output of small lymphocytes in the lymph was normal when rats were cannulated 72 hr after virus inoculation. No change was detected in the morphologic appearance of lymphocytes repopulating the blood and lymph. The deficit of lymphocytes in the blood, lymph, and lymphoid tissues following NDV challenge was similar to that produced by chronic thoracic duct drainage, nehatal thymectomy, and extracorporeal irradiation of blood (27-31). The effect of NDV on lymphocytes appeared to be selective in that anemia and granulocytopenia were not associated with virus-induced lymphocytopenia. In addition, granulocytosis was seen in all mice 12 hr after NDV challenge when the deficit of blood lymphocytes was maximal. This effect of NDV on granulocytes was markedly reduced by treating the virus with LIV irradiation or by bilaterally adrenalectomizing recipients prior to virus challenge. The procedures did not impair the ability of this virus to provoke lymphocytopenia. The mechanism(s) by which NDV alters the recirculating pool of lymphocytes is unknown. The lymphocytopenic effect of NDV could be related to its pyrogenic property (32). This does not seem likely since uv irradiation destroys NDV pyrogenicity (33) but not its ability to induce lymphocytopenia. Because the intravenous transfusion of NDV has been reported to increase plasma corticosteroid levels in mice (34), it is possible that the effect of virus on lymphocytes was mediated by stimulation of the adrenal glands (23, 35). However, the changes in the germinal centers associated with adrenal gland-mediated lymphocytopenia were not observed when NDV-challenged mice were severely lymphocytopenic. In addition, it was shown that 4 X lo8 EID,, NDV produced the same degree of lymphocytopenia in intact and bilaterally adrenalectomized mice. Virus-challenged, adrenalectomized mice also showed depletion of lymphocytes from the deep cortex of lymph nodes and from the periarteriolar lymphoid sheaths of the spleen. These results imply that a significant aspect of the effect of NDV on lymphocytes and lymphoid tissues was not mediated by the adrenal glands. In contrast, it has been reported that intracerebral inoculation of NDV resulted in destructive lesions in the thymus,

VIRUS-INDUCED

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OF LYMPHOID

TISSUES

3.51

lymph node, and spleen; these lesions were apparently mediated by the adrenal glands since they were not observed in mice adrenalectomized prior to intracerebral virus challenge (24). The fate of NDV in the host appears to be an important factor leading to the deficit of circulating lymphocytes. Procedures which restrict the distribution of NDV reduced or prevented its lymphocytopenic effect. For example, blood lymphocyte counts were normal in mice injected subcutaneously with NDV. Also, when NDV was incubated in vitro with NDV antiserum prior to intravenous transfusion, the ability of the virus to produce lymphocytopenia was significantly impaired. Previous studies have shown that this treatment increased both the rate of clearance of NDV from the blood and the accumulation of virus in the liver (21). However, it is also possible that antibody against NDV altered the surface characteristics of the virus and thereby its ability to react with lymphocytes. Nonetheless, these findings, in association with the results demonstrating a relationship between the dose of virus injected and the severity of lymphocytopenia, support the idea that lymphocytopenia was mediated by the direct interaction between lymphocytes and the virus. Preliminary observations suggest that this interaction may occur in the spleen since, in adrenalectomized mice, lymphocytopenia was not found if recipients were splenectomized 3 hr after virus challenge (Woodruff, J. J. and Woodruff, J. F.. unpublished observations). It has been shown that in recipients of tick-borne encephalitis virus, lymphocytopenia was closely correlated with the rise in titer of infective virus recovered in the blood and spleen (7). Also, although blood lymphocyte counts were not reported, severe depletion of -lymphocytes from the thymus and thymus-dependent areas of lymph nodes and spleen paralleled growth of lymphocytic choriomeningitis virus in these tissues (36). In the present experiment replication of NDV in lymphoid organs of mice could not be demonstrated as determined by recovery of infective virus in eggs. It was further shown that NDV, with no demonstrable infectivity for eggs after uv irradiation, produced lymphocytopenia and to approximately the same extent as infectious virus. These results indicate that the ability of NDV to produce infectious virus was not necessary for the production of lymphocytopenia. These observations do not exclude the possibility that this virus achieves its effect by penetrating cells of the host and initiating replication which is then aborted. In vitro studies have nrovided evidebce that noninfectious KDV can alter cell metabolism (37) and tha; infectious NDV can cause cell death without achieving a complete cycle of replication (38). Also, NDV added to lymphocytes in cultures was found to markedly impair their response to PHA (39). Thus, in the present experiments, alteration in lymphocyte metabolism could be the primary abnormality. However, modification of lymphocyte surface structures (with or without penetration of lymphocytes) by NDV may also be a critical factor in the development of lymphocytopenia. For example, neuraminidase is present on the surface of NDV (40) and evidence has been obtained that this enzyme can alter the surface characteristics and the behavior of thoracic duct lymphocytes (41) . As noted previously (32)) since most recirculating small lymphocytes are longlived (43-44), reduction in their rate of production cannot be a major factor in the rapid development of lymphocytopenia. The simplest explanation for the deficit

352

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AND

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of lymphocytes in blood and lymph after NDV challenge is that these cells were destroyed. However, procedures which cause a similar degree of lymphocytopenia by destruction of recirculating lymphocytes cause a longer period of lymphocytopenia. For example, several weeks were required for blood lymphocyte counts to return to normal levels when animals were exposed to extracorporal irradiation of blood (30)) chronic thoracic duct drainage (27)) or antilymphocyte serum (4546). Also the thoracic duct output of lymphocytes was depressed for 10 days after rats were treated with a single injection of antilymphocytic serum (47). The amount of time needed to replenish the recirculating pool of small lymphocytes is consistent with the long life-span of most of these cells. While it has been shown that in some recipients of antilymphocyte serum the duration of lymphocytopenia was brief, correction of this deficit resulted from an increased accumulation of short-lived lymphocytes in the blood (48). If, following NDV challenge, recirculating lymphocytes are destroyed then there are two apparent explanations for the rapid repopulation of blood and lymph with lymphocytes: mobilization of residual lymphocytes from lymphoid tissues and/or the entrance of large numbers of shortlived lymphocytes into these compartments. Preliminary results obtained in mice injected every 4 hr for 4 days with 3H-thymidine beginning at the time of virus challenge indicate that the majority of lymphocytes repopulating the blood at 96 hr were not newly formed cells. (Woodruff, J. J., and Woodruff, J. F. unpublished observations). Alternatively, the abrupt shifts in the concentration of lymphocytes in the blood and lymph after NDV inoculation may, in part, represent transient modification of the normal pattern of lymphocyte migration. Transient inhibition of cell-mediated immune responses has been observed in viral disease. For example, in mice, the rejection of skin allografts was delayed for approximately 2-3 days when recipients were challenged with lactic dehydrogenase virus within 4 days of transplantation (13). In man, measles virus infection frequently impairs skin tuberculin reactivity for several weeks (11). A similar, although less dramatic, depression of the tuberculin reaction has been reported following administration of viral vaccines such as measles, polio, and yellow fever ( 11, 49). The mechanisms of these phenomena are unknown. Since recirculating lymphocytes participate in several immune reactions, (SO), it may be that viruses which transiently impair the expression of cellular immunity achieve their effect by altering, as did NDV, the fate of this population of lymphocytes. The present experiments demonstrating a close association between NDV and the recirculating pool of lymphocytes may also relate to studies on the induction of interferon since data have been obtained which suggest that lymphocytes may play a role in serum interferon production in mice following NDV challenge (5 1) . ACKNOWLEDGMENTS The authors gratefully acknowledge the aid of Drs. Chandler A. Stetson, Graham H. Jeffries, Jerome L. Schulman, and Gertrude M. Schloer, and the excellent technical assistance of Miss Marjorie Dickstein and Miss Kaye Leitinger. REFERENCES 1. Benjamin, B., and Ward, S. M., Amer. J. Dis. Child. 44, 921, 1932. 2. Holbrook, A. A., Arch. Znt. Med. 66, 294, 1941.

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3. 4. 5. 6. 7. 8. 9.

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Henle, W., Henle, G., and Stokes, J., Jr., J. Immunol. 46, 163, 1943. Harris, S., and Henle, W., J. Imnzunol. 59, 9, 1948. Hillenbrand, F. K. M., Lancet 2, 66, 1956. Johnson, E. S., Napoli, V. M., and White, W. C., Amer. J. Clin. Pathol. 34, 118, 1960. Malkova, D., Pala, F., and Sidak, Z., Acta b’irol. 5, 101, 1961. Airhart, J. W., Trevino, G. S., and Craig, C. P., I. Inzmunol. 102, 1228, 1969. Hirsch, M. S., Nahmias, A. J., Murphy, F. A., and Kramer, J. H., J. Exp. Med. 129, 121, 1968. 10. Woodruff, J. F., J. Infec. Dis. 121, 164, 1970. 11. Starr, S., and Berkovich, S., N. Engl. J. Med. 270, 386, 1964. 12. Notkins, A. L., Mergenhagen, S. E., Rizzo, A. A., Scheele, C., and Waldmann, T. A., J. Exf. Med. 126, 347, 1966. 13. Howard, R. J,, Mergenhagen, S. E., Notkins, A. L., and Dougherty S. F., Transplant Proc. 1, 586, 1969. 14. Craig, C. P., Reynolds, S. L., Airhart, J. W., and Staab, E. V., J. Imnzuxol. 102, 1220, 1969. 15. Gowans, J. L., and Knight, E. J., Proc. Roy. Sot. Ser. B 159, 257, 1964. and Rickettsial Infections of Man.” 16. Schmidt, N. J., and Lennette, E. H., In “Viral (Horsfall, F. L. Jr. and I. Tamm, Eds.), p. 1212. J. B. Lippincott, Philadelphia, 1965. 17. Webster, R. G., and Pereira, H. G., J. Gen. Viral. 3, 201, 1968. 18. Ginsberg, H. S., J. Exp. Med. 94, 191, 1951. 19. Groupe, V., and Dougherty, R. M., J. Inzmzh+zol. 76, 130, 1956. 20. Ogasawara, K., and Yasue, T., Virology 6, 269, 1959. 21. Brunner, K. T., Hurez, D., McCluskey, R. T., and Benacerraf, B., J. Immunol. 65, 99, 1960. 22. Kohn, A., Virology 26, 228, 1965. 23. Dougherty, T. F., and White, A., Endocrinology 35, 1, 1944. 24. Rott, R., and Muller, G., Arch. Virusforsckung. 17, 139, 1965. 25. Gowans, J. L., Brit. J. Exp. Pathol. 39, 67, 1957. 26. Gowans, J. L., J. Physiol. London 146, 54, 1959. 27. McGregor, D. D., and Gowans, J. L., J. Exp. Med. 117, 303, 1963. 28. Parrott, D. M. V., and East, J., Nature London 195, 347, 1962. 29. Parrott, D. M. V., de Sousa, M. A. B., and East, J., J. Exp. Med. 123, 191, 1966. 30. Cronkite, E. P., Jansen, C. R., Rai, K., Cottier, H., and Fliedner, T. M., Iti “The Kinetics of Cell Proliferation.” (F. Stohlman, Jr., Ed.), p. 126. Grune and Stratton, New York, 1959. 31. Cottier, H., Cronkite, E. P., Jansen, C. R., Rai, K. R., Singer, S., and Sipe, C. R., Blood 24, 241, 1964. 32. Siegert, R., and Braune, P., Virology 24, 209, 1964. 33. Siegert, R., and Braune, P., Virology 24, 218, 1964. 34. Solomon, G. F., Merigan, T. C., and Levine, S., Proc. Sot. Exp. Biol. Med. 126, 74,1967. 35. Dougherty, T. F., and White, A., Amer. J. Anat. 77,81, 1945. 36. Hanaoka, M., Suzuki, S., and Hotchin, J., Science 163, 1216, 1969. 37. Huppert, J., Hillova, J., and Gresland, L., Natwe London 223, 1015, 1969. 38. Adams, W. R., and Prince, A. M., J. Exp. Med. 106,617, 1957. J. L., Olson, G. B., Dent, 39. Montgomery, J. R., South, M. A., Rawls, W. E., Melnick, P. B., and Good, R. A., Science 157, 1068, 1967. 40. Rott, R. In “Newcastle Disease Virus. An Evolving Pathogen.” (R. P. Hanson, Ed.), p. 133. Univ. of Wisconsin Press, Madison, 1964. 41. Woodruff, J. J., and Gesner, B. M., J. Exp. Med. 129, 551, 1969. 42. Ford, W. L., Brit. J. Exp. Pathol. 49, 502, 1968. 43. Everett, N. B., Caffrey, R. W., and Ricke, W. O., Ann. N.Y. Acad. Sci. 113, 887, 1964. 44. Robinson, S. H., Brecker, G., Lourie, I. S., and Haley, J. E., Blood 26, 281, 1965. 45. Taub, R. N., and Lance, E. M., J. Ezp. Med. 129, 1281, 1968. 46. Tyler, R. W., Everett, N. B., and Schwarz, M. R., J. Immunol. 102, 179, 1969 47. Agnew, H. D., J. Exp. Med. 128, 111, 1968.

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