Plasmodium berghei: Nucleic acid agglutinating antibodies in rats

EXPEZUMENTAL PARASITOLOGY 26, 175-180

Phmodium

berghei:

Nucleic Julius

Faculty

(1969)

Acid

P. Kreier

of Microbial

(Submitted

Agglutinating

and

Dana

Antibodies

in Rats’

A. Dilley2

and Cellular Biology, Ohio State University, Columbus, Ohio 43210 for publication

10 February

1969)

Kannm, JULIUS P., AND DILLEY, DANA A. 1969. Plasmodium berghei: nucleic acid agglutinating antibodies in rats. Experimental Parasitology 26, 175-180. The serum of rats infected with Plusmodium berghei was found to contain agglutinins for RNA and, at a lower titer, for DNA. Cross-absorption studies with RNA and trypsinized erythrocytes indicated that the anti-RNA agglutinin was not identical to the antitrypsinized erythrocyte agglutinin found in the same sera. INDEX DESCRIPTORS: Rodent

malaria,

Autoimmunity,

The course of infection in malaria is characterized by lytic destruction of erythrocytes and consequent anemia. Autoantibodies to nucleic acids have been found in association with other diseases causing destruction of host cells, including an hereditary hemolytic anemia of rats (Norins and Holmes, 1964) and systemic lupus erythematosis (Casals et al., 1963), as well as in rabbits injected with rat or rabbit liver or reticulocyte ribosomes or with yeast RNA (Bigley et al., 1963). Although antibodies to nucleic acids have not been reported to occur in animals or men with malaria, the presence of antibodies to other host cell materials has been reported. Antibodies capable of agglutinating erythrocytes in the presence of Coombs reagent have been found to occur frequently in the serum of rats infected with P. berghei (Zuckerman, 1960) and of chickens infected with P. gallinaceum I 1 This work was supported in part by ‘a grant (AI-06108-01) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service, and by the U. S. Air Force Institute of Technology. 2 Presently on active duty with the U. S. Air Force. 175

antibody

to nucleic

acids.

( Gautam, 1967), and in a few human beings infected with P. ti~ur (BarrettConnor, 1967), or P. falciparum ( Adner et al., 1968). Simple agglutination of trypsinized erythrocytes by antibodies associated with malarial infection has also been reported (Cox et al., 1965; Kreier et al., 1966). The purpose of the work reported here was to determine whether antibodies to RNA or DNA could be found in the plasma of rats infected with Plasmodium berghei. METHODS

Experimental infections. White rats of the Wistar strain, weighing 200-300 gm, were experimentally infected with the KBG173 strain of P. berghei. The strain was maintained by serial passage in stock white mice. For passage and experimental infection, blood was collected from the mice in Alsever’s solution. Three groups of rats were studied. There were minor variations in the infecting doses given the rats and in other aspects of the experimental design. Two of the rats in group I were inoculated with 0.1 ml of the infected mouse

176

KREIER

blood in Alsever’s solution; the other two rats in this group received the same volume of normal mouse blood and served as controls. The number of infected cells per dose of infected blood was not determined. Eight rats in group II received 0.3 ml of a 1: 10 dilution of the infected mouse blood-Alsever’s solution in phosphatebuffered saline (PBS). It was calculated from a dry smear count and a red cell count that each rat received about 21 million infected erythrocytes. Four control rats were given 0.3 ml of a dilution of normal mouse blood in Alsever’s solution. The infected and normal suspensions of mouse blood were compared colorimetrically, and the normal cell suspension adjusted by dilution to approximately the same density as that of the infected cell suspension. Five experimental rats in group III each received 0.2 ml of a 1:40 dilution of mouse blood-Alsever’s solution in PBS. Each rat received an estimated 5 million infected erythrocytes. Three control rats were given a comparable quantity of normal mouse blood. Hematology and serology. Blood was collected from the clipped tails of the rats by the milking technique, using as anticoagulant for each 2 ml of blood 12 units of heparin. Bleeding schedules for rats in group III are recorded in column 1 of Table I. Similar schedules were used for rats in the other groups. Blood films were made at each bleeding, stained with Giemsa stain, and examined for percentage of parasitized cells. The percentage of packed erythrocyte volume of each sample was determined by the microhematocrit technique. The remainder of the sample was centrifuged and the plasma withdrawn. The plasma was inactivated at 56°C for 30 minutes, then absorbed with washed, packed Rh + 0 human erythrocytes, using one part erythrocytes to four of plasma.

AND

DILLEY

The absorption was done to remove agglutinins for the erythrocytes. tests for antibodies to Agglutination RNA and DNA. Recently outdated Rh i- 0 erythrocytes were obtained from a Red Cross blood bank for use as “carriers” of nucleic acid. The cells were washed four times in PBS; 0.3 ml of washed, packed cells was added to each 9.15 ml of PBS. To each aliquot of erythrocyte-PBS suspension was added 0.21 ml of molar salinephosphate buffer solution containing 0.1 mg of RNA or DNA per ml. The RNA and DNA were obtained from the Nutritional Biochemicals Corporation, Cleveland, Ohio. Bis-diazotized benzidine (BDB) was used to couple the RNA or DNA to the Rh + 0 erythrocytes, and was prepared by the method of Gordon et al., (1958). Rubber gloves were used when handling BDB to protect against its cancer-producing effect. Aliquots of BDB in corked agglutination tubes were quick frozen at -78°C then stored at -70°C until used, For each experiment one tube of frozen BDB was placed in a 37°C water bath until it was just thawed. A 1:16 dilution of 0.5 ml BDB in 7.5 ml of PBS was then made. Immediately after preparation, 0.6 ml of the BDB in PBS was added to each aliquot of erythrocytes and RNA or DNA. The mixture was aIlowed to react for 10 minutes at room temperature. Then the coupled erythrocytes were packed by centrifugation at 7000 rpm for 30 seconds. The cells were washed once in a 1:lOO dilution of inactivated normal rabbit serum which had previously been absorbed with Rh + 0 erythrocytes. A 2% suspension of the coupled erythrocytes in a 1: 100 dilution of normal rabbit serum was prepared, as was a similar suspension of normal Rh -I- 0 cells which was used in control tubes. Samples of inactivated, absorbed serum from infected and control animals were serially diluted

NUCLEIC ACID AGGLUTINATING ANTIBODIES

with PBS in agglutination tubes in dilutions ranging from 1:2 to 1:64. Control tubes for each series of tests contained PBS only, or a known positive serum, or a normal serum. The known positive reactor with RNA was serum from a rabbit which had been injected with liver ribosomes 63 days prior to sample collection. The known positive for DNA was undiluted serum from a human with lupus erythematosus. The normal control for RNA was normal rabbit serum; for DNA, normal human serum. To each 0.4-ml volume of serially diluted serum or control serum was added 0.1 ml of a suspension of DNA or RNA coupled to erythrocytes. The same quantity of the control suspension of normal erythrocytes was added to aliquots of each control serum and to 1:4 dilutions of the test sera. The tubes were incubated at 37°C for 30 minutes and centrifuged 15-30 seconds at full speed, then examined for presence or absence of agglutination. Cross-absorption agglutination tests. For these tests a pool was made of sera from P. berghei-infected rats which had been shown to contain antibodies for RNA and the antibody for trypsinized rat erythrocytes reported earlier (Kreier et al., 1966). The serum was absorbed with Rh + 0 human erythrocytes for 25 minutes at 37°C. The serum was then divided into four aliquots; one of these was absorbed with trypsinized rat erythrocytes, one with RNAcoupled human erythrocytes, one with normal rat erythrocytes, and one was not absorbed. Serial twofold dilutions of 1:%1:64 of each of the four aliquots of serum in PBS were prepared in 0.2 ml quantities in agglutination tubes. Two tenths milliliter of rat erythroa suspension of trypsinized cytes, the preparation of which was described by Kreier et al. ( 1966), was added to each tube, and the tubes examined for agglutination as described by Kreier et a2. ( 1966). After reading of tests for agglutina-

177

tion of trypsinized rat erythrocytes, the tubes were stored at 4°C for 12 hours to allow the cells to settle out. The supernatant serum was then transferred to another set of tubes and 0.1 ml of RNAcoupled human erythrocytes added to each tube. After appropriate incubation the tubes were examined for agglutination of RNA-coupled cells. RESULTS

Tests for agglutinins of RNA and DNA. Serum from all but one of the P. bergheiinfected rats agglutinated RNA-coupled erythrocytes. The exception was an animal in group II which did not show parasitemia until the fifth day after infection, and died on that day. Of sera from P. berghei-infected rats tested for agglutinins of DNAcoupled erythrocytes (those in groups II and III), all but two showed positive reactions at some stage of the infection. One of the exceptions was the nonreactor with RNA mentioned above; the other was an animal which died on the fifth day after infection without ever showing patent parasitemia, but which showed agglutinins for RNA on the fifth day. Sera from uninfected control rats never agglutinated RNA or DNA-coupled erythrocytes. The highest titer obtained for positive reaction with RNA was 1:64; serum from all but two rats reached a titer or at least 1:8 on at least one day. The highest titer obtained with DNA was 1: 16; serum from all but the two nonreactor rats reached a titer of at least 1:8 on at least one day. Of the 11 rats whose serum showed agglutinins for both RNA and DNA, 9 developed a higher maximum titer for RNA than for DNA, one developed a higher maximum titer for DNA, and one developed the same maximum titers for both. The agglutinin for RNA was often first found earlier in the infection than that for DNA. Table I shows the presence of the agglutinms in rats of

I78

KREm

AND DILLEY TABLE

I

Hematology and Antibody to Nucleic Acids in Nom1 Plasmodium Per cent infected cells AVa

Days Noninfected -5c -1 1 3 6 8 10 Infected -5 -1 1 3 6 8 10 a b c d e

SDb

Per cent packed cells

Rats and in Rats Infected with

berghei Anti-RNA (units/O.2 ml)

Per cent reticulocytes

AV

SD

AV

SD

AV

SD

0 0 0 0 0 0 0

49.6 45.2 41.5 42.3 41.3 39.5 38.8

.647 1.0 .289 .440 .670 1.266 .728

.43 .97 6.0 5.3 11.6 9.7 13.3

.068 .200 1.156 .647 ,872 1.196 1.428

--d -

0 0 0 0 0 0 0

0 0 4.109 1.182 4.175 0

48.8 53.0 44.6 42.5 33.7 23.9 20.3

1.137 .659 1.188 1.358 3.918 1.378 1.238

3.2 1.7 4.2 9.4 11.3 38.2 40

1.571 1.348 .933 2.633 2.214 5.194 7.002

-

0 0 1.262 5.657 6 0

Anti-DNA (units/O.4 ml) AV

SD

rats 0 0 0 0 0 0 0

. . .e ... ... ...

... 0 . . 0 . . 0 ...

rats 0 0 4.02 22.7 30.5 24

Average for three normal or five infected Standard deviation. - indicates days before injection. - indicates no agglutination detectable. . . . indicates not tested.

4 16 22 32

...3.3 2.4 5 10.5 8

0”’ 3.194 1.597 1.917 3.402 0

rats.

group III during the course of infection and blood sampling. The average units of antibody listed are calculated as averages of the reciprocals of the titers for each serum sample tested on a given day. Results for tests with the other two groups of rats were similar, except that no DNA tests were run for Group I. Relation of test results to course of infection. All control rats showed some decrease in packed cell volume and increase in reticulocytosis during the period of bleeding, but anemia and reticulocytosis were more marked in the infected rats. One of the two infected rats in group I survived; the other died on Day 17 after inoculation. All the rats in group II died, one on Day 5, two on Day 8, three on Day 9, one on Day 10, and one on Day 11 after inoculation. The five rats in group III died, on Days 5, 8, 9, 11, and 13 after inocula-

tion. Table I shows average parasitemias, packed cell volumes, and reticulocytosis for the rats in group III through the course of the study. Positive agglutination titers occurred only when parasitemia was evident, and high titers tended to occur when parasitemia was high. Values for parasitemia, packed cell volume, and reticulocytosis for rats in the other two groups were similar to those shown for group III, and tests for these rats showed correlations between parasitemia and agglutination titers similar to that shown for group III. Cross-absorption tests. Results of tests run on two pools of serum are shown in Table II. Sera unabsorbed or absorbed with normal rat erythrocytes had titers against both trypsinized erythrocytes and RNA-coupled erythrocytes. Absorption with trypsinized erythrocytes abolished the reaction with trypsinized erythrocytes but

NUCLEIC

TABLE

Cross-Absorption Studies and to Typsinized Type of absorption Pool I Unabsorbed serum Absorbed Tr-RBC Absorbed RNA Absorbed normal rat RBC Pool II Unabsorbed serum Absorbed Tr-RBC Absorbed RNA Absorbed normal rat RBC

ACID

AGGLUTINATING

II of

Antibodies to RNA Eythrocytes

Titers against trypsinized rat erythrocytes

Titers against RNA

1:32 1:16

1:16 1:32 -

1:16

1:32

1:16 1:16

1:4 1:32 -

1:16

1:32

not that with RNA. Absorption with RNA abolished the reaction with RNA-coupled erythrocytes, but reaction still occurred with trypsinized erythrocytes. DISCUSSION AND CONCLUSIONS

The negative results of all tests on uninfected controls, and the high correlation between parasitemia and positive agglutination tests in P. berg&-infected animals point strongly to the conclusion that agglutinins for RNA and DNA are produced in rats as a result of the malarial infection, and are not caused by bleeding for the tests or other extrinsic factors. It can be questioned whether the test results actually indicate the production of two agglutinins specific, respectively, for RNA and DNA. The structural similarity of the nucleic acids is such that an antibody for one might react with the other. Because the reactions with RNA often began earlier in the course of disease, and usually reached a higher titer than those with DNA, it seems likely that the agglutinins are produced in response to RNA and cross-react with DNA. Plasmodium berghei preferentially infects, and subsequently

179

ANTIBODIES

destroys, reticulocytes. The destruction of reticulocytes releases ribosomes, rich in RNA, into the bloodstream. The consequent presence of large quantities of RNA in the bloodstreams of Plasmodium berghei-infected rats could trigger the production of antibody to RNA. However, as nucleated erythrocyte precursors in the bone marrow or peripheral circulation of malarious animals are sometimes destroyed, thus releasing DNA, the possibility of two specific agglutinins cannot be dismissed. Antibodies to nucleic acids could play a role in recovery from malarial infection by aiding in the disposal of debris from destroyed parasites and destroyed erythrocytes. However, such antibodies could also cause pathologic processes. It is, for example, possible that anti-RNA antibody might bind to and destroy erythrocytes with adsorbed nucleic acids. Anti-RNA antibody might also be involved in damage to kidneys. Kidney damage associated with malaria in humans has frequently been reported; the possible immunological basis of this damage is discussed in a report on immunology of malaria (WHO, 1968). Ward and Conran (1966), using fluorescent antibody staining, found y and PlC globulins in association with what they assumed to be malarial debris in the renal glomeruli of monkeys infected with P. cynomolgi. The role of antibodies to nucleic acids in the physiology and pathology of malariainfected animals remains to be clarified. REFERENCES ADNER, M. M., ALTSTATT, L. B., AND CONRAD, M. E. 1966. Coombs’-positive hemolytic disease in malaria. Annals of Inter& Medi-

cine 68, 33-38. BARRETT-CONNOR, E. 1967. Plasmodium vivar malaria and Coombs-positive anemia. American Journal of Tropical Medicine and Hygiene 16, 699-702. BIGLEY, N. J., DODD, M. C., AND GEYER, V. B. 1963. The immunologic specificity of anti-

180

KREIER AND DILLEY

bodies to liver ribosomes and nuclei. Journal of Zmmuno.?ogy 90, 416-423. CASALS, S. P., FRIOU, G. J., AND TEAGUE, P. 0. 1963. Specific nuclear reaction pattern of antibody to DNA in lupus erythematosus sera. Journal of Laboratory and Clinical Medicine 62, 625-631. Cox, H. W., SCHROEDER, W. F., AND RISTIC, M. 1965. Erythrophagocytosis associated with anemia in rats infected with Plasmodium berghei. Journal of Parasitology 51, 35-36. of erythrocyte GAUTAM, 0. P. 1967. Detection bound globulin in Plasmodium gallinaceuminfected chickens. Ph.D. dissertation, Ohio State University. GORDON, J., ROSE, B., AND SEHON, A. H. 1958. antibodies Detection of “non-precipitating” in sera of individuals allergic to ragweed pollen by an in vitro method. Journal of Experimental Medicine 108, 37-51.

KREIER, J., SHAPIRO, H., DILLEY, D., SZILVASSY, I. P., AND RISTIC, M. 1966. Autoimmune reactions in rats with Plasmodium berghei infection, Experimenta Parasitology 19, 155162. NOHING, L. C., AND HOLMES, M. C. nuclear factor in mice. Journal ogy 93, 148-154.

1964.

Anti-

of Zmmunol-

WARD, P. A., AND CONRAN, P. R. 1966. Immunopathologic studies of simian malaria. Military Medicine 131 (suppl. to No. 9), 1225-1232. of Malaria.” World W.H.O. 1968. “Immunology Health Organ. Tech. Rep. Ser. No. 396, pp36-37. ZUCKERMAN, A. 1960. Autoantibody with Plasmodium berghei. Nature 190.

in rats 185, 189-