Indian Journal of Experimental Biology Vol. 38, Apri I 2000, pp. 313-322

Correlation and modulation of Ehrlich ascites tumor growth with tumor-plasma IgA

A K Ghosh & S Saha*

Department of Metabolic Regul ation, Chittaranj an National Cancer Institu te, 37, S.P. Mukherjee Road, Calcutta 700 026, India

Received 21 April 1999: revised 27 December / 999

Plasma IgA level of Ehrlich asc ites tumor bearing mice showed correlation with progress of tumor growth. In PAGE analys is total plasma IgA separated into 3 major bands corresponding to mol. wt. 2669,000 daltons, = 443 ,000 daltons and bet ween 443,000 and 150,000 daltons. All the three bands increased gradually with progress of tumor growth upto day 14 and then declined on day 16. Total plasma IgA isolated by anti -IgA affinit y chromatography when adopti vely transferred to mice inhibited tumor growth. Affinity- purified plasma

. IgA separated into three major peak fractions after Sephadex G-200 column chromatography which corresponded with the bands of lgA on PAGE analys is. Three Sephadex G-200 IgA fractions when adopti vely transferred to tumor-bearing mice showed effect different from total IgA. High mol. wt. lgA fracti on (2 669,000 daltons) inhibited tumor growth whereas medium mol. wt. fraction (= 443,000 daltons) enhanced tumor growth . The low mol. wt. IgA frac tion (<443,000 and > 150,000 daltons) had no significant effect on tumor growth . The high mol. wt. lgA frac tion enhanced tumor killing ability of peripheral blood lymphocytes (PBL) and peritoneal macrophages of tumor bearer in vitro. Medium mol. wt. IgA fraction inhibited tumor-killing ability of PBL in vitro but had no significant effect on peritoneal macrophages. The low mol. wt. IgA fraction showed a mild enhancing effec t on tumor-killing abi lity of PBL but had no significant effect on peritoneal macrophages. The results established importance of lgA in tumor growth regulation and its therapeutic potenti ality. The results indicated that tumor growth modulation by tumor plasma IgA is also mediated by its effect on cellular anti ­tumor immune fac tors of the host.

When a tumor develops in a host it is recognized as foreign by the immune machinery of the host due to presence of tumor-associated and/or tumor-specific antigens on its surface. The recogniti on is expressed by the generati on of both humoral , i.e., anti-tumor antibodi es and cellular components like cytotoxic killer T-lymphocytes, macrophages against the tumor. The humoral immune system of a host, speciall y IgG and IgM type of antibody against tumor-associated and/or tumor-spec ific anti gens have been establi shed to p·lay a vital role in tumor protecti ve mechani sm. IgG as well as IgM type of antibody have been reported in many investigations to give protection to a host against tumor growth 1' 4. whereas in some other studies these immunoglobulins, spec ially IgG type of antibody have been shown to enhance tumor growth5

.8

. However, there are very few reports regarding role of IgA in cancer. In fact, in case of most antigens in animals as well as humans the

*Correspondent author

recogmtton as foreign is first expressed by the generation of IgM type of antibodies by developi ng lymphoid cells of the plasma cell line (B-cell s). Then depending on the nature of antigens the antibody production switchovers to IgG class . The ce ll s committed to synthesis of IgA type antibody also ari se from the cell s that formerl y made IgM type of antibody by genetic switchover mechanism following the sequence IgM~ IgG~ IgA. Antibodies against EAC cell s are expected to follow similar sequence.

IgA type of antibody has been suggested to inhi bit growth of tumor ce ll s under in vitro cultu re conditions9

•1 1

• Serum IgA leve ls have been shown to increase in human patients with cancer of oral

. . b d h t 2- t 9 B . cav tty , cervtx , reast an nasop arynx . n ese et. al. 14

• have shown that concentrati on of secretory IgA in circulation of patients with breast cancer increased significantly . In fac t, IgA type of antibody predominates in secretory mucosal and glandul ar sites, but from these sites come to the circulation through lymphatics. There is onl y one report

314 INDIAN J. EXP. BIOL., APRIL 2000

showing tumor-growth regulatory role of IgA in • v • l ?QO d h vtvo; o1son et a . - reporte t at growth of sarcoma

in Ic mice could be enhanced by treatment with antigen-antibody complex of IgA. Although all these studies indicated that besides IgG and IgM, IgA type of ant ibody has some definite role in malignancy , but this role of IgA has not been studied so far in detail. Therefore in the present investigation with Ehrlich ascites tumor model, role of tumor-plasma IgA in tumor growth regulation and therapeutic implication, if any , of this immunoglobulin class has been evaluated .

Materials and Methods Tumor-Ehrlich ascites tumor maintained by serial

transplantation in adult male Swiss mice by intraperitoneal inoculation of I xI 06 viable tumor cells/mouse, was used.

Plasma-The plasma were separated by centrifugation from heparinized blood of normal as well as Ehrlich ascites tumor bearing mice on day 2, 4, 6, 8, I 0, 12, 14 and 16 after tumor cell inoculation, ip, on day 0.

Preparation of anti-lgA affinity Column-Mouse anti-IgA antibody (Sigma Chern. Co., St. Louis) tested to be specific for IgA by immunogel diffusion in agar plate against standard mouse IgA, IgG and lgM was used for the preparation of anti-IgA affinity column .

Freeze-dried powder of CNBr-activated Sepharose 4B (Sigma Chern. Co., St. Louis; 1.5 g) was swollen for 15 min in 5 ml of I mM HCI and washed on a sintered glass filter (porosity G3) with 300 ml of the same HCI solution. The gel was then washed with I 0 ml of 0.1 M NaHC03 coupling buffer, pH 8.3, containing 0.5 M NaCl, suspended in 7.5 ml of the same buffer and immediately transferred to a stoppered glass tube containing 14 mg anti-IgA antibody in 2.5 ml of NaHC03 coupling buffer and mixed in an end-over-end fashion for 2 hr at room temperature. After 2 hr the gel was poured into the sintered glass filter, washed with I 0 ml of 0.1 M tris­HCI buffer, pH 8, a blocking buffer, suspended in the same buffer and kept for 2 hr at room temperature. Thus the remaining active groups in the gel were blocked. To remove the excess unabsorbed protein, if any, the gel was washed alternatively with high and low pH buffer solutions four times . Sodium acetate buffer, 0.1 M, pH 4 and NaHC03 coupling buffer pH 8.3, each containing 0.5 M NaCl were used for this purpose. The unreacted blocking agent was washed away with NaHC03 coupling buffer. Anti-lgA

coupled gel was powred into a small column, equilibrated with PBS, pH 7.2 and used for affinity separation of IgA from plasma.

Separation of plasma !gA by anti-lgA affinity column-Immunoglobulin A (IgA) from I ml plasma of normal mice and from I ml plasma, each of Ehrlich ascites tumor bearing mice on day 2, 4, 6, 8, I 0, 12, 14 and 16 were separated by anti-IgA affinity column . Each I ml plasma diluted to 3 ml with PBS, pH 7 .2, was added stepwise ( I ml at a time) to the column, allowed to be absorbed for 15 min each time and then the column was washed thrice with 10 ml PBS, pH 7.2 to remove unabsorbed proteins . The bound IgA from affinity column was eluted in a 3 ml fraction with glycine-HCI buffer, pH 2.5 to a total volume of 24 mi. The fractions were pooled and immediately dialysed against I 00 ml PBS, pH 7.2 for 24 hr with two changes of the dialysing medium. The total volume of the lgA solution was reduced to 0.8 ml and again dialysed agai nst I 00 mi. PBS, pH 7.2 for 24 hr with 4 changes of the dialysing medium. Then the volume of the lgA solution was adjusted to I ml with PBS, pH 7.2, kept below -2. '°C until further analysis.

Polyacrylamide gel electrophoretic (PAGE) analysis of lgA-Affinity purified lgA of normal plasma as well as of tumor-plasma on different days of tumor growth were analysed by 7.5% PAGE using 0.2M tris-glycine, pH 8.5 as electrophoresis buffer at a constant current of 30 rnA. The gel was si lver stained following the method of Bloom et al.21

.

Standard proteins of known mol. wt. (thyroglobulin: mol. wt. 669,000 daltons, apoferritin : mol.wt. 443,000 daltons, alcohol dehydrogenase: mol. wt. 150,000 daltons) were run parallel in the gel to ascertain the moL wt. of the lgA bands.

Quantitation of lgA bands separated on PAGE using a gel scanner- Silver-stained lgA bands on PAGE were scanned at 410 nm using a Shimadzu CS-9000 Scanner. The lgA bands separated according to their different mol. wt. were scanned and recorded as different peaks. The peaks as well as the peak areas were printed out by the printer. These peak areas were proportional to the amount of lgA present in the band which indirectly represented the concentration of that lgA in the plasma.

Adoptive tram.fer of fgA in mice with tumor cell or after tumor cell inoculation-Affinity purified tumor pl asma lgA in PBS, pH 7.2, was divided into two parts. One part was mixed wi th Ehrlich tumor cel ls in PBS , pH 7.2 and injected, ip, to each

GHOSH & SAHA: TUMOUR GROWTH MODULATION BY IMMUNOGLOBULIN A 3 15

mou se of a group of 4 mice on day 0, so th at each

mouse rece ived 250 11g of lgA and I x I 05 tumor cells in 0.5 ml PBS. A second group of 4 mice received I x I 05 tumor ce ll s/mouse, ip , in 0 .5 ml

PBS on day 0 and 250 11g lgA in I ml PBS/mouse, ip, on day I , 2 and 3 from the other part of lgA solution . The control group received only I x I 0 ·4

tumor cells/mouse, ip , on day 0. Another group of mice received lgA purified from normal mou se plasma in a similar manne r as in group I. Thi s was lgA control for group I . On day 4 after tumor inoculation tumor cells were washed out from the peritoneal cavity of each mou se with 5 ml PBS , pH 7.2. The tumor ce ll count/ml was determined by trypan blue dye exclusion technique and the total count was obtained by multipl yi ng the count/ml with volume.

Separation of affinity purified tumor plasma lgA by Sephadex G-200 column chromatography-Affinity purified tumor-plasma IgA separated into three peak fraction s ( I, 2 and 3) when passed through a Sephadex G-200 column, equilibrated and eluted with PBS, pH 7.2 into 5 ml fractions. Three peak fractions I, 2 and 3 corresponded with the mol. wt. standards , thyroglobulin (669,000 daltons) , apoferritin (443,000 daltons) and alcohol dehydrogenase ( 150,000 daltons) respectively run in the same column . Three fraction s gave precipitin bands against anti-IgA antibody in immunogel diffusion study.

Adoptive transfer of Sephadex G-200 lgA-fractions in mice after tumor cell inoculation- Three groups of

mice received lx I 05 tumor cells/mouse, ip, in 0.5 ml PBS on day 0. Then these three groups received Sephadex G-200 IgA-fractions I, 2 and 3 respectively in various doses, ip , on day I, 2 and 3. The control

group received only I x I 05 tumor cells/mouse, ip, on day 0. Tumor cell counts of each mouse of each group were taken on day 4 using trypan blue dye exclusion technique.

51 Cr-release assay to study the in vitro tumor­killing by peripheral blood lymphocytes ( PBL) and peritoneal macrophages-Peripheral blood lymphocytes (PBL) were separated from heparinized blood (diluted I: I with RPMI-1640 medium) of day-6 tumor bearing mice by centrifugation over Fiquol Hypaque layer at 400 g for 30-40 min at 18°-20° C. The buffy coat of lymphocyte at the interface was removed with a pasteur pipette, washed thrice with 5 volumes of medium, counted in a haemocytometer and diluted to the desired concentration with medium (+10% FBS) .

Peritoneal macroph ages were collected from day-6-tumor-bearing mice by washing with heparinized PBS , pH 7.2 and centrifuged . The cells were suspended in 5 ml RPMI-1640 (+I 0% FBS ) medium and immediately tran sferred to a petri-di sh and incubated for I hr at 37°C in a 5% COr incubator. The supernatant poured off, added 5 ml PBS containing I o·4 M EDTA and incubated at room temperature for I 0 min . The cells were di s lodged by gentle scraping with EDT A-PBS, pH 7 .2 and finally suspended in RPMI-1640 (+I 0% FBS) medium.

Ehrlich tumor cells collected from asc ites fluid of Ehrlich ascites tumor-bearing mice were washed thrice with PBS , pH 7 .2, then twice with RPMI-1640 medium (+I 0% FBS) and adjusted to a concentration

of 2x I 06/ml. From this suspension I ml was labelled

with 300 11ci of 51Cr for 2 hr in a C02-incubator with occasional mixing, centrifuged at 800g for I 0 min and washed 5 times with RPMI-1640 medium to remove unlabeled 51 Cr. Finally the cells were counted and kept at 4°C .

To I 0 111 of lgA so lution ( 1.5 nmol e of each fraction) in each well of a microtitre plate was added 5x I 03 51 Cr-labe led tumor cells in 50 111 RPMI-1640 medium (+10 % FBS) and incubated for 30 min at 30° C. Then 5x l05 1ymphocytes or macrophages in 50 111 RPMI-1640 complete medium was added. Additional medium was added to make the volume up to 150 111 , centrifuged at room temperature for 3 min at 80 g to facilitate

cell contact; then incubated for 4 hr at 37°C in a

C02-incubator. Finally added 50 111 ice cold PBS and centrifuged at 400 g for I 0 min at 4 °C. From

eac h well I 00 111 supernatant was carefully removed, transferred to Bio-vi a l and counted in a gamma counter. Cent per cent release was determined by adding 50 111 of I % triton x-1 00 solution to the cell suspension (5x I 03

) in medium . The percent killing was dete rmined using the following formula :

5 'Cr- label released in the experimental 100 Per cent killing=

51 x

I 00% Cr - released

Count of the experimenal

-Count due to spontaneous release x 100

Count due to Triton X- I 00 treatment

-Count due to spontaneous release

Statistical significance was determined by Student 's t

test in all the experiments

316 INDIAN J. EXP. BIOL., APR IL 2000

Results Changes in plasma lgA with progress of Ehrlich

ascites tumor growth-Affinity purified plasma JgA on different days of tumor growth, e.g., day 2, 4, 6, 8, I 0, 12, 14 and 16, as analysed by PAGE has been presented in Fig. I. PAGE analysis revealed that plasma JgA of Ehrlich tumor-bearing mice separated into 3 major bands (I, II and ITT). When these bands were compared with standard mol. wt. markers like thyroglobulin (mol wt. 669,000 daltons), apoferritin (mol. wt. 443,000 daltons) and alcohol dehydrogenase (mol. wt. 150,000 daltons) run parallel to these lgA samples, indicated that band I had mol. wt. ~ 669,000 daltons, band II mol wt. around 443,000 daltons and band Ill mol. wt. between 443,000 and 150,000

11

lll

daltons . Moreover, stain ing intensity of the lgA bands suggested quantitative changes i111 all the three types of JgA bands with progress of tumor growth .

The concentration of plasma IgA bands on polyacrylamide gel were determined by scanning with a gel scanner. The concentrations of different mol. wt. IgA on different days of tumor growth as expressed by di fferent peak profiles have been presented in Fig. 2. The quantitative data as calculated from the peak areas for different mol. wt. IgA on di ffe rent days of tumor growth have been presented in Fig. 3. Both Figs 2 and 3 confirmed the visual observation on PAGE. Total plasma IgA of day 2 tumor bearing mice was 1.13 times higher than that of normal plasma and the level gradually increased with progress of tumor

669000

44 300 0

2 '· 6 8' 10 12 14 16 s

Fig. 1-Arli nity purified pl as ma lgA on different days of tumor growth, e.g., day 2, 4 , 6, 8, 10, 12, 14 and 16. as analysed by polyacrylamide gel (7.5 %) electrophoresis (PAGE) using 0.2 M Tri s-glyci ne, pH 8.5 as electrophoresis buffer at a constant cu rrent of 30 mA and si lver stained. Standard proteins of known mol. wt. , e.g., thyroglobulin (mol.wt. 669,000 daltons), apoferrit in (mol. wt. 443 ,000 daltons) and alcohol dehydrogenase (mo l. wt. 150,000 dal tons) were run parallel to ascertain the mol. wt. of the lgA bands separated on PAGE (S); N: lgA from normal mouse plasma.

4.070

~ E 3.270

0 :; t-<l: 2.4 70 >- · t: Ill z UJ 1.670 0 _J

<l: u t- 0.870 n. 0

0.00 0 NORMAL DAY 2 4 6 8 10 12 14 16 PLASMA I lgA TUMOR PLASMA lgA--------...J

•

Fig. 2-Concentration profiles of plasma-l gA of different mol. wt. , separated on PAGE for different days of tumor growth , as scanned by a gel scanner at 41 0 nm.

GHOSH & SAHA: TUMOUR GROWTH MODULATION BY IMM UNOGLOBU LI N A 3 17

growth. On day 14 of tumor growth the level became almost 3.5 times the normal value and subsequentl y decreased on day 16 to a level of 2.44 times the normal va lue. The levels of three major IgA bands in the tumor plasma alsc, increased graduall y with progress of tumor growth . The levels were maximum on day 14 of tumor growth , about 2.6 times the normal va lue fo r IgA of mol. wt. ;::: 669,000 daltons, 4

times for IgA of mol. wt. = 443 ,000 daltons and 4 .2 times for IgA of mol.wt. between 443,000 and 150,000 daltons. The leve ls of high , medium and low mol. wt. IgA came down to a value of 1.6, 2.5 and 3.4 times the normal value respective ly on day 16 of tumor growth .

Effect of adoptively transferred tumor plasma lgA on Ehrlich ascites tumor growth-When affinity purified pl asma IgA of Ehrlich asci tes tumor-bearing mice was adoptive ly transferred wi th tumor cells ( lx l05/mouse) to normal mice on day 0, ip , and the tumor ce ll counts were taken on day 4, the number of tumor ce ll s were found to decrease significantl y (55%, P < 0.01) compared to control mice, receiving only tumor cel ls on day 0 (Table I). Thi s inhibitory effect was dose dependent and the maximum effect

obtained at 250 !lg of IgA has been presented in Table I. In another group of mice receiving tumor

r-en 0

0 • enM .,;

~ M ~. <: N N

In

~ en

"" ;:! CDN en .

M .~ 0 M <nM ~. ;: ..,,.; "" a> "! en N

or- r--.._: "1~

nnn

cell s on day 0, ip , and tumor plasma IgA, ip, on day I , 2 and 3, tumor cell counts were also observed to

decrease significantly (56%, P < 0 .0 I) compared to contro l (Table I). Thi s was also a dose dependent

effect and the max imum effect was obtained at 250 !lg of IgA. The control group receiving IgA from normal mouse plasma showed tumor cells counts almost same as that in control group receiving only tumor cells (Table I, Expt. 2), i.e., normal mouse plasma IgA had no effect on Ehrlich asc ites tumor growth.

Effect of adoptively transferred tumor-plasma lgA of different mol. wt. on Ehrlich ascites tumor growth-Affinity purified total IgA of tumor-plasma was separated by Sephadex G-200 column chromatography into its vari ous mol. wt. fractions as shown in Fig. 4 . Three peak fraction s (Frac. I , Frac. 2 and Frac. 3) were obtai ned wh ich corresponded with the standard mo l. wt. markers thyroglobulin (660,000 daltons), apoferritin (443,000 daltons) and alcohol dehydrogenase ( 150,000 da ltons) respective ly, run in the same co lumn. Three fracti ons were tested for their effect on Ehrlich tumor growth. The fractions showed effect diffe rent from total plasma IgA. The hi gh mol wt. fraction (;::: 669,000 daltons) when injected , ip, on day I, 2, and 3 after tumor cell inoculation on day 0, ip , inhibited tumor

M 0 0

IDM-.i NO"'"1 ID ~. "'! r"!

N ~ N en~ -: ("') PJ . -:N -t.~

M ~ cnN NN N N ~ · "!

U'lU'lN_!

""

nnnn nTI NT 2r 41 61 81 1Df1q 14r16r N L 2 L 4 L 6 L 8L10L1 2L 14L 16L NM 2M 4t.i 6M 6t.1 1ty 21J~16M ~2H ~ ~8H 1(\1~11-!16H

1- To tal pt asma l gA~ rlow mol.wt.lgA 4 ~M4!dium mo l.wt. lgA_, ~High mol.wt.lgA.f

Fig. 3-Plasma lgA levels on different days of Ehrlich ascites tumor growth. 2T, 4-r •........... 16T represent total plasma lgA on respective days of tumor growth . 2L, 4L·········· · 16L; 21\-, 4M· ···· ······· 16M and 2~" 4H··········· 16H represent concentration of low, medium and hi gh mol. wt. plasma IgA respectively on respective days of tumor growth .Bar heights are directly proportional to the peak areas of the respective lgA bands on polyacrylamide gel, scanned by a Gel-scanner, which in tern are proportional to the concentration of the respecti ve lgA in plasma. NT. NL, NM and NH represent total, low mol. wt., medium mol. wt. and high mol. wt. lgA of normal mouse plasma. NL, NM and NH are 31.5, 29.0 and 39.5 percent respectively of NT as determined from their scanned peak area values . Number on each bar means corresponding normal bar va lue multipli ed by that number represent that particular bar value. Bar height of NT =30 mm (arbitraril y chosen). Results of one from two representati ve experiments have been presented.

318 INDIAN J. EXP. BIOL., APRIL 2000

growth significantly (55 %, ?<0.0005 ) and the inhibition was dose dependent. The maximum inhibition was obtained at 250 11g IgA/mouse (Table 2). The lgA of medium mol wt. (= 443000 daltons) enhanced tumor growth in a dose dependent manner and the maximum effect was obtained at 250 11g IgA/mouse (39%, P < 0.0005). However IgA of low mol. wt. (443,000-150,000 daltons), showed no effect on tumor growth (Table 2).

Survival of the group receiving high mol. wt. IgA was 100% on day 22 when all the mice of control were dead (data not shown).

Effect of d(fferent mol. wt. plasma-lgA of Ehrlich ascites tumor-bearing mice on the tumor killing ability of peripheral blood lymphocytes (PBL) and peritoneal macrophages of the same tumor-bearer in vitro-The tumor killing ability of PBL and peri toneal macrophages of Ehrlich tumor-bearing mice was studied in vitro by 51 Cr-release assay in presence of high mol. wt. IgA-fraction (~ 669,000 daltons), medium mol. wt. IgA-fracti on (=443,000 daltons) and low mol. wt. IgA fraction (443,000-150,000 daltons), obtained from Ehrlich tumor­plasma by Sephadex G-200 column chromatography . The high mol. wt. IgA-fraction added at a dose of 1.5 nmole enhanced tumor-k illing ability of PBL by approximately 161 %, whereas the same dose of medium mol. wt. IgA-fraction inhibited tumor-k illing

abi li ty of PBL by 32.5%. The low mol. wt. JgA­fraction enhanced tumor-killing abil ity of PBL only by 25% (Table 3).

The high mol. wt. IgA-fraction added at a dose of 1.5 nmole enhanced tumor-killing ab ility of peritoneal macrophages by approximately 130%. The medium mol. wt. IgA-fraction at the same close inhibited tumor-killing ability of macrophages only by 3% which was not stati stically significant. Similarly low mol. wt. IgA-fraction enhanced tumor-killing ability of macrophages only by 5.5% which was also not statistically significant (Table 3). The dose of 1.5 nmole was the optimum dose to show maximum effect in case of both PBL and peritoneal macrophages. Normal plasma-IgA showed no effect on PBL and peritoneal macrophages (data not shown).

Discussion Using Ehrlich ascites animal tumor model in the

present in vestigation it has been establi shed that there is definitely a correlation between plasma IgA level and progression of tumor growth. Plasma IgA level gradually increased upto day 14 of tumor growth, then decreased on day 16 when the animals began to die due to tumor burden. Similar type of correlation between tumor growth and plasma IgA level was observed in studies with 3-methylcholanthrene induced fibrosarcoma bearing C3H/HeJ mice22

. Al so plasma IgG and IgM levels of fibrosarcoma-bearing

Table 1-Effect of adoptively transferred tumor-plasma IgA on Ehrli ch ascites tumor growth Treatment

Received I x l 05 tumor cell + 250 J..lg lgA from tumor pl as ma/mouse, ip, on day 0

Received I X I 05 tumor cell s/mouse, ip, on day 0 and 250 J..l g lgA from tumor plasma/mouse, ip, on day I, 2 & 3

Received lx l05 tumor cells + 250J..! g lgA from normal mouse pl asma, ip, on day 0

Received on ly Jx J05 tumor cell s/mouse, ip, on day 0 (control ).

D =not done; NS =not significant

Expt. o. Count of tumor cell s (x i O.~) on day 4 after Tumor

2 3

2 3

2 3

2 3

tumor cell inocul ation on day 0. inhibition [Mean±SD of 4 tumor cell counts from (%)

4 mi ce for each group in each experiment]

38.5±14.7 19.6±2.9

NO

29.4±11.3 24.3±2.3 48.0±0.9

NO 46.7±4.16

NO

78.4±11.4 47.8±9.9

99.6±2 1.5

50.9 58.9

62.5 49.0 56.0

Significance ? -values

<0.01 <0.01

<0.005 <0.01 <0.01

NS

GHOSH & SAHA: TUMOUR GROWTH MODULATION BY IMM UNOGLOBU LIN A 319

Table 2-Effect of tumor-plasma IgA of different mol. wt. on the growth of Ehrlich ascites tumor

Treatment : lgA injected i.p. on day I , 2 and 3 to groups of mice receiving tumor cells ( Ixl05/mouse, ip) on day 0.

Hi gh mol. wt. lgA-fraction

(~ 669,000 daltons)

Medium mol. wt. lgA- fracti on (= 443,000 daltons)

Low mol. wt. lgA-fraction (443,000 to 150,000 daltons)

No lgA injected . Received only I xI 05 tumor cells/mouse, i.p. on day 0 (Control)

Group

I.

2. 3.

I.

2.

I. 2.

c

Doses OflgA

(Jlg)

250

ISO 50

250

ISO

250 ISO

Tumor cell count(x I o·6) on day 4 after Tumor cell inocu lat ion on day 0.

[Values are mean± SD of 4 tumor cell counts from 4 mice for­each group. Figures in the parentheses for groups of high mol. wt. lgA denotes percent .inhibition, for medium mol. wt. lgA

denotes percent enhancement and for low mol. wt. IgA denotes percent inhibition]

Expt. I 20.67±3.63(5 1.0)

22.10 ±2.40(46.0) 22.93±2.27(45.5)

56.80±3.81 (34.6)

50.53±2.81 ( 19.7)

41.40±5 .31(1.9) 42.17±1.40(0.3)

42.20±2.39

Expt. 2 8.50±3 .52 (65.2)

15.75±1.55(35.5) ND

36.25±5.24(48.6)

29.40±4.04(20.5)

ND ND

24.42±2.54

Expt. 3 23 .04±1 .27(50.5)

33.20±3.82(28.6) 36.20± 1.44(22.2)

61 .82±2.59(32.9)

54.43±2.48( 17 . I)

ND ND

46.56±1.80

Signi licance P-values

<0.0005 for Expt.l , 2&3

<0.0005 for Ex pt. I, 2&3

<0.0005 for Expt I &3

<0.0005 for Expt I &3

<0.005 for Expt 2 <0.005 for Expt

1&3 <0.05 for Expt 2

NS NS

ND =not done ; NS =not significant.

Table 3-Effect of different mol. wt. pl asma lgA-fractions of Ehrlich tumor-bearing mice on the tumor killing abili ty of peripheral blood lymphocytes (PBL) and peritoneal macrophages of the same tumor-bearer studied in vitro by 51Cr-release assay

Group No.

No. of tumor cells/ o. of lymphocytes

I. Sx 103/Sx l 05 ( I: I 00) 2. Sx I 03/Sx I 05 ( I : I 00)

3. Sx I 03/Sx I 05 ( I :I 00)

4. 5x l03/5x l 05 ( 1:1 00)

No. of tumor cell s/ No. of macrophages

I. Sx I 03/Sx I 05 ( I : I 00) 2. 5x 103/5x l05 (1:100)

3. Sx 103/Sxl 05 ( 1:1 00)

4. Sx I 03/Sx 1 05 ( I : 1 00)

Sephadex G-200 Percent tumor killing Percent Enhancement (En) or Signifi-lgA fractions [Values are mean± S.D. of Inhibition (In) in tumor killing by cance added 4 separate determinations. lgA fractions P-values ( 1.5 nmole) Counts (c. p.m.) for I 00%

release was I 000 ± I 0 and for spontaneous release was

180 ± 5] Expt. I Expt. 2

Nil 21.68±0.58 12.47±2.02 Frac I (tetrameric 44.80±1.61 39.5 1±1.47 and/or higher polymeric-lgA) Frac 2 (trimeri c- 14.45±4.20 8.50±0.69 lgA) Frac 3 (di-and/or 24.R5± 1.76 16.84±3.87 monomeric-lgA)

Ni l 23.73±5.78 I 1.26±2.26 Frac I (tetrameric 40.94±3.93 32.39±2.74 and/or hi gher polymeric lgA) Frac2 (trimeri c- 23. 12±4.93 10.78±4.89 lgA) Frac 3 (di-and/or 24.77±0.60 12.04±2.27 monomeri c-! A)

Expt. 1 Expt. 2

106.6 (En) 216.8(En) <0.0005, <0.0005

33.3 (In) 3 1.8 (1n) <0.0 1, <0.005

14.6 (En) 35 .5(En) <0.01 , <0.05

72.5 (En) 187.6(En) <0.0005, <0.005

2.5 (In) 4.2( 1n) NS, s

4.4 (En) 6.9(En) NS , NS

320 INDIAN J. EXP. BIOL., APRIL 2000

mice increased with progress of tumor growth compared to normal control mice but did not show any linear correlation with tumor growth22

. Therefore in the present in vestigation with Ehrlich asc ites tumor model we became interested about plasma IgA level but not plasma IgG or IgM levels . Moreover, it has been shown in the present investigati on that IgA, obtained from the plasma of Ehrlich asc ites tumor­bearing mice when adoptively transferred to normal mice with tumor cell s or after tumor cell inoculation could inhibit subsequent tumor growth significantl y (55-56%). So far tumor inhibitory antibodi es have been shown to be of IgG or IgM type 1

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Appokrritia ( •ol. wt. 443 ...

40 60 60 100

FRACTION NUMBER

Fig. 4-Separation of affini ty purified tumor-p lasma lgA by Sephadex G-200 column chromatography. Tumor-plasma lgA separated into three peak fractions ( I, 2 and 3) when passed th rough a Sephadex G-200 column, equili brated and eluted wi th PBS, pH 7.2, into 5 ml fractions . Three peak fractio ns, I , 2 & 3 corresponded wi th the mol. wt. standards thyrogl obulin (669,000 daltons), apoferritin (443,000 daltons) and alcoho l dehydrogenase ( 150.000 daltons) respectively, run in the same column.

for the first time in the present study, it has been shown that IgA type of antibodies may have si mil ar effect. In the present study normal mouse plasma IgA did not show any tumor growth inhibitory effect, suggesting that tumor-growth inhibitory effect is possibly due to the presence of tumor-spec ific IgA­type antibody in IgA preparation obtained from tumor-plasma. This results as well as resul ts reported

. ~ h . 9-1 1 15-1 9 h I A wtt1t uman cancer pattents · suggest t at g level in the circulation may be a good marker for diagnosis as well as prognosis of tumor.

The more interesting observation is that while total plasma IgA transferred adoptively inhibited tumor growth, IgA-fract ions separated from tumor-plasma on the basis of mol. wt. acted differently. High mol. wt. IgA-fracti on (~ 669,000 daltons) inhibited tumor growth whereas med ium mol. wt. IgA-fraction (= 443,000 da ltons) enhanced tumor growth . The low mol. wt. IgA-fraction (between 443,000 and 150,000 daltons) had no effect on tumor growth. Taking into cons ideration the mol. wt. of monomeric IgA as 160,000 daltons, the high mol. wt. fraction is suggested to be tetramer and/or higher polymers of IgA, the medium mol. wt. fraction to be trimeric IgA and the low mol. wt. fracti on to be dimeric and/or monomeric IgA .

At present it is not sure why tetra and/or hi gher polymers of IgA and trimeric IgA are exerting tota ll y opposite effect on tumor growth and what are their mechanism of action. However, in one mechani sm it was suggested that tumor escapes immune destruction by the host by suppressing anti-tumor immune

f I h b bl k. "b d" 5-8?4 ?5 responses o t 1e ost y oc mg ant1 o tes ·- ·- . So, the medium mol. wt. IgA-fraction, i.e. trimeri c IgA, showing tumor enhancement in vivo, may be blocking antibodies. Th is view is supported by the observation that trimeric IgA-fraction of tumor­plasma inhibited tumor--ki lling abil ity of PBL and peritoneal macrophages of tumor-bearer in vitro. In another mechanism it was suggested that tumor escapes immune destructi on by the host by suppressing the immune reaction of the host against the tumor. This occurs through blocking factors which are suggested to be tumor-specific and/or tumor­associated antigens. Tumors constantly shed such anti gens into the circulation which form complexes with the tumor-specific antibodies and thus precipitate the anti-tumor antibodies in the ci rculation before they can reach the tumor cell s and are el iminated from the circulation in due course5

. Simi lar mechani sm may be operative in tumor specific IgA molecules

GHOSH & SAHA: TUMOUR GROWTH MODULATION BY IMMUNOGLOBULIN A 321

present in the circulation of EAC-bearing mice. They are precipitated in the circulation before they can reach the intraperitoneal cavity. On the other hand, the IgA molecules adoptively transferred with EAC cells are directly coming in contact with the tumor cells and in combination with macrophages and/or lymphocytes are killing the tumor cells before they are precipitated by the antigens shed by the tumor cells. The high mol. wt. IgA-fraction , i.e., tetrameric and/or, higher polymeric IgA-fraction showing tumor growth inhibition in vivo, may be anti-tumor antibody. Moreover, thi s tetrameric and/or, higher polymeric IgA-fraction enhanced tumor-killing ability of PBL and peritoneal macrophages of tumor-bearer in vitro, suggesting that anti-tumor effect of thi s high mol. wt. IgA-fraction is also mediated by enhancing the tumor­killing ability of cellular anti-tumor factors of the host. Role of low mol. wt. lgA-fraction , i.e. , di­and/or, monomeric forms of IgA in vivo is not clear, although its level in the circulation increased with progress of tumor growth and thi s fraction of IgA showed mild enhancing effect on the tumor-killing ability of PBL in vitro but had no significant effect on peritoneal macrophages. Possibl y thi s low mol. wt. IgA-fraction has some additive effect to the tumor­killing ability of PBL, besides hi gh mol. wt. fgA­fraction.

In conclusion it is worth mentioning that quite opposite effect of IgA of different mol. wt. on tumor growth indicates that proper manipulation of these biomolecules may have potential therapeutic application. However, before considering therapeutic application, above information should be established with etiologically defined solid animal tumor models. Besides its therapeutic application IgA may also have its diagnostic as well as prognostic application. In fact, Iglehart et.a/. 26 establi shed correlation between secretory 1mmune system, namely IgA and bronchogenic carcinoma and suggested that measurement of secretory IgA may be useful in the earl y diagnosis of malignant bronchogenic di seases and they also suggested clinical application of lgA. Similarly, Khalifa et.a/. 27 suggested use of IgA as tumor-markers in bladder cancer of human s. Zong et.al?8 suggested use of IgA antibody against viral capsid antigen of Epstein-Barr virus (EBV) for screenmg of early stage of nasopharengeal carcinomas, as they observed the presence of this antibody in the blood of many subjects during screening of a population in southern China. Petrelli et.al. 29 demonstrated the clinical utility of serum IgA-

I as a potential complementary tumor markers to CEA in the monitoring of the post-operative course of patients with advanced colorectal adenocarcinoma.

Acknowledgement Thanks are due to Dr. Putul Maity , Department of

Metabolic Regulation and to Professor M A Siddiqi , Director for co-operation and encouragement.

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