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DNA content of lymphocytes in adjuvant arthritis of rats
Exp. Pathol. 30, 243-246 (1986) VEB Gustav Fischer Verlag Jena 1) Research Institute of Rheumatology, Prague; 2) Institute of Pharmacology, Czechoslovak Academy of Sciences, Prague; 3) Research Institute of Pharmacology and Biochemistry, Prague
Short communication
DNA content of lymphocytes in adjuvant arthritis of rats
With one figure Address for corresponde11:-ce: Dr. BLANKA Sr
Introduction
Measurement of DNA content in cell nuclei is at present widely used as a relatively reliable marker for the assessment of biological behaviour of tumors. In diagnostics of non-tumorous diseases the quantitative microscopical methods are used less frequently but they are source of valuable information on the response of the organism to various noxae. FCA-induced arthritis in rats has been a suitable model of an inflammatory disease for decades (DuMoNDE et al. 1977). The current understanding of pathogenesis of adjuvant arthritis is mainly immunological suggesting an impairment in T-lymphocyte function (TAUROG et al. 1983). The kinetics of participating cells are known only from few sources (BONVOISIN et al. 1984) and therefore we tried in the present study to determine and quantify the effect of FCA upon immunocompetent cells, namely lymphocytes . .11aterials and Alethods 10 male Wistar rats (weighing on avemge 100 g) were used. 5 animals served as controls whereas 5 rats received a single subcutaneous dose of FCA (0.4 ml/kg). The adjuvant was prepared from 1.3 mg of inactivated Mycobacterium tuberculosis 1/47 dissolved in 1.0 ml of paraffin oil. 30 days after FCA administration the rats were sacrificed and peripheral blood smears together with imprints of spleen and sternum were prepared. By this time, macroscopically evident signs of arthritis were developed. The slides were stained with May-Griinwald-Giemsa method and for fluorometry a modified Feulgen technique of NAVARETTE et al. (1983) was applied. The fluorescence-emission intensity of Feulgen-stained nuclei (given in arbitrary units, AU) was measured with a Leitz MPV 2 photometer equipped with epifluorescence illuminator Ploemopak 2 and with HBO 100 lamp as a UV light source. The photometer was interfaced to a Hewlett Packard 85 desk computer for data retrieval, storing and further processing. 150 nuclei in each slide were measured using rectangular measuring diaphragm 12 X 12 jtm at wavelength 580 nm. The data stored on tape were processed with original HP statistical program ONESAM. The significance of difference between means of control and experimental measurements was computed with HP program TSTAT2. The DNA index (DI) was calculated from the ratio of cells in Gi/o peak of experimental animals to Gi/o peak of control peripherallymphocytes. Results
The administration of FCA resulted in an increase of circulating mature granulocytes accompanied with relative fall in lymphocytes number (table 1). The mean fluorescence emission of Feulgen-stained DNA in lymphomonocytoid cells in the control sternal imprints increased after FCA from originally 47.52 AUto 223.58 AU (table 2). The same trend was observed in the spleen and peripheral blood but to a lesser extent. The changes in fluorescence intensity were statistically significant (p < 0.01) when compared against controls. 16*
243
NO % 63 42 56 49
37. 32. 28
35 28 21 1-4
23. 18. 14 9.3 4.6
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21. 18. 16 13.
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Fig. 1. Distribution of the DNA content in blood, spleen and bone marrow lymphocytes from rats with adjuvant arthritis in comparison to blood lymphocytes from control animals. Abscissa: fluorescence intensity in arbitrary units (AU) Ordinate: number and percentage of cells. Table 1. Differential counts of blood smears of control and FCA-treated rats. Average values of 5 rats are shown
Granulocytes segmented rods Eosinophils Lymphocytes Monocytes
Control
FCA
13 0.3 1 84.7 1
50.6 2.6 2 41 3.8
Peripheral lymphocytes of control animals displayed the diploid (2C) population at 130 AU (fig. 1). FCA-treatment resulted in the shift towards polyploidy, most prominent being in the sternum and spleen while in the blood the majority of lymphocytes displayed a 3C state of ploidy. DNA index was higher than 1.0 in all organs pointing thus to an increase of proliferation activity of lymphocytes (table 3). Discussion
FCA similar to other derivatives of bacterial walls affects the immune system. The administration of a classical adjuvant induced appearance of T-cell subpopulations that participated in initiation of inflammatory joint disease (TAUROG et ai. 1983). This finding is in
244
Exp. Pathol. 30 (1986) 4
~
>I>Ot
t-:)
II>-
~
~
~ ~
~
"d
'?
± 17.43
150.27
± 12.89
116.67
157.02 ± 15.49 148.34 ± 14.98 145.36 ± 17.8G 143.93 ± 20.89 156. 70 ± 17.94
105.47 ± 14.80 116.79 ± 9.42 122.78 ± 9.59 118.94 ± 12.80 119.37 ± 17.85
± 11.70 ± 12.78 ± 15.10 ± 15.50 ± 11.80 101. 76 ± 13.38
Control
Control 104.55 99. 81 101.36 95.28 107.78
Spleen
F CA
Peripheral blood
± 46.10 ± 40.1 7 ± 37.08 ± 37.07 ± 34.81 203.43 ± 39.05
211.8G 207.12 207.32 195.76 195.11
FCA
47.52
± 10.76
44.68 ± 9.92 45.27 ± 11.82 49.05 ± 10.97 47.22 ± 9.64 51.37 ± 11.49
Control
St ernum
Values obtained in experimental animals differed statistically from controls (t-test fo r means of 2 samples, p < 0.01)
Mean
1. 2. 3· 4. 5.
Rat No.
223.58
± 54.04
219. 19 ± 38.98 217.41 ± 58.79 23 7.96 ± 59.42 217.70 ± 59.15 225.66 ± 53.84
FCA
Table 2. Fluorometry of DNA in ly mphomonocytoid cells in peripheral blood smears, spleen and sternal i mprin ts. The fl uorescen ce in tensity and standard deviations are given in arbit rary units (AU)
Table 3. DNA index (DI) of control and experimental animals. Peripheral lymphocytes were used as 2C population
Bone marrow Spleen Blood
Control
FCA
0.44 1.04 1.00
1.73 1.67 1.39
agreement with our observation of the stimulation of proliferation activity of lymphocytes and lymphomonocytoid cells especially in the bone marrow. We were not able to differentiate between subsets of participating cells but we presume that regulation of the lymphomonocytoid cell-line maturation is one of the primary effects of FCA. Moreover, we deal here with a longlasting effect as well, since we have observed the prominent changes even 30 days after administration of the initiating agent. Similar effects were observed with other drugs with immunoregulatory potential (JULls et al. 1985), suggesting that these compounds find at least some of their target cells in the bone marrow. Elevated DNA synthesis rate in peripheral blood lymphocytes can then be explained by an increased influx of less mature elements from the site of origin. At present, lymphocytes and macrophages are favored as target cells of various immunomodulating drugs (CHEDID et al. 1983; LECLERC and CHEDID 1982). The dynamics of their DNA kinetics in this connection is known from a limited number of sources only (LECLERC and CHEDID 1982; GALLELI and CHEDID 1983; JULIS et al. 1985). An increase in the number of mature granulocytes in peripheral blood may be considered a secondary reaction in FCA induced inflammation. From our results it seems that systemic administration of FCA in doses high enough for development of adjuvant arthritis influenced the DNA synthesis rate even one month later. As expected, the brunt of changes was detected in the bone marrow suggesting the promoting effect of FCA upon production, maturation and efflux of lymphocytes and monocyte-like cells.
Literature BONVOISIX, B., CORDIER, G., REVILLARD, J. P., LEJEUNE, E., BOUVIER, M., Increased DNA and/or RNA content of synovial cells in rheumatoid arthritis: a flow-cytometry study. Ann. Rheum. Dis. 1984; 43: 222-227. DUIVIONDE, D. C., JONES, E. H., KELLY, R. H., OATES, C. M., Experimental models of rheumatoid inflammation. Bayer-Symposium VI, Experimental models of chronic inflammatory diseases. Springer-Verlag, Berlin-Heidelberg-New York, 1977, pp. 4-27. CHEDID, L., AUDIBERT, F., JOHNSON, A. G., Biological activities of muramyl dipeptide, a synthetic glycopeptide analogous to bacterial immunoregulating agents. Prog. Allergy 1983; 25, 63-105. GALLELI, A., CHEDID, L., Modulation of myelopoiesis in vivo by synthetic adjuvant active muramyl peptides: Induction of colony-stimulating activity and stimulation of stem cell proliferation. Infect. Immunitv 1983; 42: 1081-1085. JULIS, I., KADLECO~A, 0., l\L>\SEK, K., HORNIKOVA, M., Quantitative analysis of muramyl dipeptide effect on rat macrophages. Histochem. J., in press. LECLERC, C., and CHEDID, L., Macrophage activation by synthetic muramyl peptides. Lymphokines, Academic Press, London-New York 1982, Vol. 7, pp. 1-21. NAVARETTE, M. H., SECADES-CAMPOS, E., HURTADO, M. S., Analisis citofotometrico de las conditiones optimes de la tintion de Feulgen. Morfol. Normal. Pathol. 1983; 7: 239-247. TAUROG, J. D., SADBERG, G. P., MAHOWALD, M. L., The cellular basis of adjuvant arthritis. Cell. Immunol. 1983; 80: 198-204. (Received March 11, 1985)
246
Exp. Pathol. 30 (1986) 4
Short communication
DNA content of lymphocytes in adjuvant arthritis of rats
With one figure Address for corresponde11:-ce: Dr. BLANKA Sr
Introduction
Measurement of DNA content in cell nuclei is at present widely used as a relatively reliable marker for the assessment of biological behaviour of tumors. In diagnostics of non-tumorous diseases the quantitative microscopical methods are used less frequently but they are source of valuable information on the response of the organism to various noxae. FCA-induced arthritis in rats has been a suitable model of an inflammatory disease for decades (DuMoNDE et al. 1977). The current understanding of pathogenesis of adjuvant arthritis is mainly immunological suggesting an impairment in T-lymphocyte function (TAUROG et al. 1983). The kinetics of participating cells are known only from few sources (BONVOISIN et al. 1984) and therefore we tried in the present study to determine and quantify the effect of FCA upon immunocompetent cells, namely lymphocytes . .11aterials and Alethods 10 male Wistar rats (weighing on avemge 100 g) were used. 5 animals served as controls whereas 5 rats received a single subcutaneous dose of FCA (0.4 ml/kg). The adjuvant was prepared from 1.3 mg of inactivated Mycobacterium tuberculosis 1/47 dissolved in 1.0 ml of paraffin oil. 30 days after FCA administration the rats were sacrificed and peripheral blood smears together with imprints of spleen and sternum were prepared. By this time, macroscopically evident signs of arthritis were developed. The slides were stained with May-Griinwald-Giemsa method and for fluorometry a modified Feulgen technique of NAVARETTE et al. (1983) was applied. The fluorescence-emission intensity of Feulgen-stained nuclei (given in arbitrary units, AU) was measured with a Leitz MPV 2 photometer equipped with epifluorescence illuminator Ploemopak 2 and with HBO 100 lamp as a UV light source. The photometer was interfaced to a Hewlett Packard 85 desk computer for data retrieval, storing and further processing. 150 nuclei in each slide were measured using rectangular measuring diaphragm 12 X 12 jtm at wavelength 580 nm. The data stored on tape were processed with original HP statistical program ONESAM. The significance of difference between means of control and experimental measurements was computed with HP program TSTAT2. The DNA index (DI) was calculated from the ratio of cells in Gi/o peak of experimental animals to Gi/o peak of control peripherallymphocytes. Results
The administration of FCA resulted in an increase of circulating mature granulocytes accompanied with relative fall in lymphocytes number (table 1). The mean fluorescence emission of Feulgen-stained DNA in lymphomonocytoid cells in the control sternal imprints increased after FCA from originally 47.52 AUto 223.58 AU (table 2). The same trend was observed in the spleen and peripheral blood but to a lesser extent. The changes in fluorescence intensity were statistically significant (p < 0.01) when compared against controls. 16*
243
NO % 63 42 56 49
37. 32. 28
35 28 21 1-4
23. 18. 14 9.3 4.6
42
7 0
HO
COHTROL
~
BLOOD
21. 18. 16 13.
32 28 24 211
16 19. 12 B 5.3 8 4 2.6 0
LIMIS>
HO
lSI
lSI
lSI
lSI
iii
~.,
lSI lSI N
lSI iii (\J
(II
lSI M
~
(II
on·
!-)
CIo .,;)
&
In -t
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on
19 8 6
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4 2
os'
lSI
is' .,;)
.,;)
is'
b")
.,;)
N
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~.~ NO 16 1e. 14 9.3 12 B
STERHOH
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OS) (II If)
Fig. 1. Distribution of the DNA content in blood, spleen and bone marrow lymphocytes from rats with adjuvant arthritis in comparison to blood lymphocytes from control animals. Abscissa: fluorescence intensity in arbitrary units (AU) Ordinate: number and percentage of cells. Table 1. Differential counts of blood smears of control and FCA-treated rats. Average values of 5 rats are shown
Granulocytes segmented rods Eosinophils Lymphocytes Monocytes
Control
FCA
13 0.3 1 84.7 1
50.6 2.6 2 41 3.8
Peripheral lymphocytes of control animals displayed the diploid (2C) population at 130 AU (fig. 1). FCA-treatment resulted in the shift towards polyploidy, most prominent being in the sternum and spleen while in the blood the majority of lymphocytes displayed a 3C state of ploidy. DNA index was higher than 1.0 in all organs pointing thus to an increase of proliferation activity of lymphocytes (table 3). Discussion
FCA similar to other derivatives of bacterial walls affects the immune system. The administration of a classical adjuvant induced appearance of T-cell subpopulations that participated in initiation of inflammatory joint disease (TAUROG et ai. 1983). This finding is in
244
Exp. Pathol. 30 (1986) 4
~
>I>Ot
t-:)
II>-
~
~
~ ~
~
"d
'?
± 17.43
150.27
± 12.89
116.67
157.02 ± 15.49 148.34 ± 14.98 145.36 ± 17.8G 143.93 ± 20.89 156. 70 ± 17.94
105.47 ± 14.80 116.79 ± 9.42 122.78 ± 9.59 118.94 ± 12.80 119.37 ± 17.85
± 11.70 ± 12.78 ± 15.10 ± 15.50 ± 11.80 101. 76 ± 13.38
Control
Control 104.55 99. 81 101.36 95.28 107.78
Spleen
F CA
Peripheral blood
± 46.10 ± 40.1 7 ± 37.08 ± 37.07 ± 34.81 203.43 ± 39.05
211.8G 207.12 207.32 195.76 195.11
FCA
47.52
± 10.76
44.68 ± 9.92 45.27 ± 11.82 49.05 ± 10.97 47.22 ± 9.64 51.37 ± 11.49
Control
St ernum
Values obtained in experimental animals differed statistically from controls (t-test fo r means of 2 samples, p < 0.01)
Mean
1. 2. 3· 4. 5.
Rat No.
223.58
± 54.04
219. 19 ± 38.98 217.41 ± 58.79 23 7.96 ± 59.42 217.70 ± 59.15 225.66 ± 53.84
FCA
Table 2. Fluorometry of DNA in ly mphomonocytoid cells in peripheral blood smears, spleen and sternal i mprin ts. The fl uorescen ce in tensity and standard deviations are given in arbit rary units (AU)
Table 3. DNA index (DI) of control and experimental animals. Peripheral lymphocytes were used as 2C population
Bone marrow Spleen Blood
Control
FCA
0.44 1.04 1.00
1.73 1.67 1.39
agreement with our observation of the stimulation of proliferation activity of lymphocytes and lymphomonocytoid cells especially in the bone marrow. We were not able to differentiate between subsets of participating cells but we presume that regulation of the lymphomonocytoid cell-line maturation is one of the primary effects of FCA. Moreover, we deal here with a longlasting effect as well, since we have observed the prominent changes even 30 days after administration of the initiating agent. Similar effects were observed with other drugs with immunoregulatory potential (JULls et al. 1985), suggesting that these compounds find at least some of their target cells in the bone marrow. Elevated DNA synthesis rate in peripheral blood lymphocytes can then be explained by an increased influx of less mature elements from the site of origin. At present, lymphocytes and macrophages are favored as target cells of various immunomodulating drugs (CHEDID et al. 1983; LECLERC and CHEDID 1982). The dynamics of their DNA kinetics in this connection is known from a limited number of sources only (LECLERC and CHEDID 1982; GALLELI and CHEDID 1983; JULIS et al. 1985). An increase in the number of mature granulocytes in peripheral blood may be considered a secondary reaction in FCA induced inflammation. From our results it seems that systemic administration of FCA in doses high enough for development of adjuvant arthritis influenced the DNA synthesis rate even one month later. As expected, the brunt of changes was detected in the bone marrow suggesting the promoting effect of FCA upon production, maturation and efflux of lymphocytes and monocyte-like cells.
Literature BONVOISIX, B., CORDIER, G., REVILLARD, J. P., LEJEUNE, E., BOUVIER, M., Increased DNA and/or RNA content of synovial cells in rheumatoid arthritis: a flow-cytometry study. Ann. Rheum. Dis. 1984; 43: 222-227. DUIVIONDE, D. C., JONES, E. H., KELLY, R. H., OATES, C. M., Experimental models of rheumatoid inflammation. Bayer-Symposium VI, Experimental models of chronic inflammatory diseases. Springer-Verlag, Berlin-Heidelberg-New York, 1977, pp. 4-27. CHEDID, L., AUDIBERT, F., JOHNSON, A. G., Biological activities of muramyl dipeptide, a synthetic glycopeptide analogous to bacterial immunoregulating agents. Prog. Allergy 1983; 25, 63-105. GALLELI, A., CHEDID, L., Modulation of myelopoiesis in vivo by synthetic adjuvant active muramyl peptides: Induction of colony-stimulating activity and stimulation of stem cell proliferation. Infect. Immunitv 1983; 42: 1081-1085. JULIS, I., KADLECO~A, 0., l\L>\SEK, K., HORNIKOVA, M., Quantitative analysis of muramyl dipeptide effect on rat macrophages. Histochem. J., in press. LECLERC, C., and CHEDID, L., Macrophage activation by synthetic muramyl peptides. Lymphokines, Academic Press, London-New York 1982, Vol. 7, pp. 1-21. NAVARETTE, M. H., SECADES-CAMPOS, E., HURTADO, M. S., Analisis citofotometrico de las conditiones optimes de la tintion de Feulgen. Morfol. Normal. Pathol. 1983; 7: 239-247. TAUROG, J. D., SADBERG, G. P., MAHOWALD, M. L., The cellular basis of adjuvant arthritis. Cell. Immunol. 1983; 80: 198-204. (Received March 11, 1985)
246
Exp. Pathol. 30 (1986) 4