Nephron 23 : 293-2% (1978)

Effect of Renal Physicochemical Milieu on Stimulation of Human Lymphocytes by Phytohemagglutinin1 Herbert J. Harwich. George M. Kalmanson andLucien B. Guze Research and Medical Services, Wadsworth Veterans Administration Hospital; Department of Medicine, UCLA School of Medicine, Los Angeles, Calif, and Department of Medicine, Harbor General Hospital, Torrance, Calif.

Key Words. Renal milieu • Lymphocyte • Phytohemagglutinin

It has previously been shown that the unique physico­ chemical environment of the kidney interferes with normal serum bactericidal activity [1] and phagocytosis [2]. One of the major pathologic changes in pyelonephritis is mono­ nuclear cell infiltration. It is probable that these cells are functioning immunologically. It was therefore of interest to determine the effect of this milieu on T cell lymphocyte function as exemplified by ability to be stimulated by phytohemagglutinin. We have found that a variety of factors do interfere with such stimulation.

Materials and Methods Preparation o f Homan Lymphocytes Normal human volunteers were used. Approximately 35 ml of peripheral blood were drawn into a syringe containing 0.36 ml of 6% dextran (Sigma, St. Louis. Mo.. MW 260,000) and 0.05 ml of 1% heparin (Calbiochem, San Diego. Calif.). After 30 min sedimentation at room temperature, the supernatant plasma containing white cells was aspirated and centrifuged at 400 g for 5 min. The lymphocytes were not further purified as PHA stimulates principally lymphocytes only. The cells were resuspended in medium 199 containing Hank's balanced salt solution and glutamine (Gibco, Grand Island, N.Y.) with 10% fetal calf serum (Gibco), 1% Hepcs buffer 2.5 M (Calbio­ chem) and 1% antibiotic-antimycotic solution (Gibco). 1 Supported in part by VA Medical Research and N1H Grant Sup­ port A 102257.

Stimulation Test The test with human lymphocytes was done as follows: lympho­ cyte viability was determined by trypan blue (Baker Chemical Co., Phillipsburg. N.J.) exclusion. Cells were always -95% viable. After one wash 5 x |0 15viable cells were dispensed into 17 x 100 mm plastic culture tubes (Falcon, Oxnard, Calif.). PHA-M (Difco, Detroit, Mich.), 0.25 ml, was added to each tube, volume adjusted to 10 ml with culture medium and cells were uncubated at 37 C. The PHA solution as made up was 1% in saline giving a final concentration of 400 gg/ml. This concentration is a little higher than that used by others [3], However, this amount was found to give excellent stimu­ lation, giving 20,000-30.000 cpm as compared to about 125 cpm in unstimulated control cultures. At the end of 72 h of incubation, an aliquot of cells was removed for redetermination of viability. After 72 h cultures were centrifuged at 400 g for 5 min, washed and resus­ pended to 3 ml with culture medium. I gCi tritiated thymidine — 3 HtdR (Amersham & Searle, Des Plaines, III.) (specific activity 5 Ci/mmol) was added and cultures further incubated for 3 h at 37 C. 3 ml of 10% trichloracetic acid (TCA) were then added. The cultures were centrifuged at 1,200 g for 5 min, washed twice with 2.5 ml of 5% TCA. 0.4 ml of 0.5 A’NaOH and I drop of 30% hydrogen peroxide (Mallinckrodt, St. Louis. Mo.) were added to the precipitate. After overnight at 4 C, 0.1 ml was added to a scintillation vial followed by 0.2 ml water and 9 ml scintillation fluid. The scintillation fluid contained 50% Instagel (Packard, Downers Grove. III.) and 50% toluene with 46 mg 5-phenyloxazolylbenzene (Packard) and 4 g 2,5diphenyloxazol (Sigma) per liter toluene. Vials were refrigerated at 4 C in the dark overnight and counted. Results were recorded as counts per minute per vial (CPM). Controls included cultures without PHA-M n = 138. 125.67 ±4.66 (mean ; standard deviation of mean). Cultures without 3 HtdR had negligible activity.

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Abstract. The effect of changes in certain physicochemical parameters, analogous to those occurring in the kidney, on stimulation of normal human lymphocytes by phytohemagglutinin was studied. Increase in osmolality by sucrose or urea, and increases in concentration of sodium, potassium and calcium all significantly inhibited stimulation while magnesium did not. Lowering pH to 6.8 and 6.5 inhibited stimulation. Raising the pH to 8.0 had no effect but at pH 8.4 stimulation was decreased. The effect was largely but not entirely due to decrease in viability.

294

Harwick/Kalmanson/Guze

Tabic I. Effect of individual renal physicochemical parameters on stimulation of lymphocytes by phylohcmagglutinin CPM2

Parameter tested1

Significance1

concentration per liter

osmolality mosm/kg

experimental

control3

Sucrose

80 m M 150 mM 260 mM

375 450 600

14,017 £ 4,015 ± 59 £

854 350 5

23,754 £1,416 30,232 £ 1,659 22,958 £ 2,766

S S S

Sodium

183 mEq 228 mEq

375 450

13,494 £ 2,762 £

951 229

23,754 £ 1,416 30,232 1,659

S S

44 mEq 94 mEq

375 450

5,784 £ 170 £

989 19

21,941 £1,349 20,263 £ 1,592

S S

80 mM 160 mM 220 mM 280 mM

375 450 535 600

17,270 £ 1,596 14,334 £ 1,199 15,793 £ 1,056 251 £ 36

21,941 £ 20,263 £ 29,627 £ 22,958 £

1,349 1,592 1,678 2,766

S S S S

15 mEq

340

15,927 £ 1,244

22,850 £ 1,208

S

312

20,224 £ 2,956 £ 14,094 £ 25,628 £ 23,791 £

22,850 £ 26,254 £ 30,269 £ 26,254 £ 30,269 £

NS S

Potassium Urea

Calcium Magnesium pH 6.5 6.8 8.0 8.4

-

-

-

-

-

-

-

1,619 705 1,460 2.420 1,774

1,208 2.305 2,308 2,302 2,308

s NS

s

5 replicates from 5 individuals tested, thus 25 numbers for each test. Counts per minute per tube after 72 h incubation. Mean £ SD of mean. Basic medium contains: sodium 147 mEq, potassium 6.5 mEq, calcium 1.6 mEqand magnesium 0.45 niEq. The osmolality is 310. pH is 7.25. Experimental vs. control by T test. S = 0.05 or better.

Modification o f Cultare Medium Osmolality was increased by adding sucrose or urea. Sodium, potassium, calcium and magnesium were increased by adding the chloride salt, taking into account the composition of the basal medi­ um. pH was altered by adding either sodium hydroxide or hydro­ chloric acid. The modifications used were based on those of Chernew and Braude [2] and Lamdin [4]. The figure for magnesium was kindly supplied by Dr. Shaul Massry.

Results The results are shown in table I. Each number repre­ sents the average of 5 replicate samples from each of 5 individuals. For statistical purposes the 25 individual values were used. Addition of sucrose to increase osmo­ lality from baseline 310 mosm/kg; 80 mM sucrose, 375 mosm/kg; 150 mM sucrose, 450 mosm/kg; and 260 mM. sucrose 600 mosm/kg all significantly inhibited stimulation. Addition of urea to reach the same osmolalities also in­ hibited stimulation as compared to unmodified control,

but significantly less so than did sucrose. Sodium, 183 mEq, 375 mosm/kg; 228 mEq, 450 mosm/kg; decreased stimu­ lation but only to the same extent as sucrose at similar osmolalities. On the other hand, potassium, 44 mEq, 375 mosm/kg; and 94 mEq. 450 mosm/kg. inhibited stimulation significantly more than could be accounted for by increase in osmolality. Calcium 15 mEq, 340 mosm/kg significantly reduced stimulation about the same as sucrose while magnesium 1.5 mEq. 312 mosm/kg was inactive. Reducing pH from baseline 7.2 to 6.8 and 6.5 significantly reduced the response. Raising the pH to 8.0 had no effect while further increase to 8.4 slightly but significantly reduced stimulation. All modalities tested except magnesium 1.5 mEq, 312 mosm/kg, and pH 8.0 significantly reduced stimulatability as indicated by decreased counts per minute as compared to the unmodified control. Question then arises as to whether this is due simply to loss of viability. Data on viability are shown in table II.

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1 2 3 1

1.5 mEq -

Renal Milieu and Lymphocytes

295

It should be noted that initially viability ofall white cells was determined. At 72 h only lymphocytes and monocytes remained viable. This probably accounts for the drop in present viability of the controls to about half. Those para­ meters which decrease further the viability were affecting the lymphocytes. Magnesium did not significantly reduce viability (table II) nor did it inhibit stimulation (table I). At pH 6.5, the viability was significantly higher than the control and yet stimulation was significantly reduced (table I). At pH 8.0, viability was significantly reduced, yet stimulation was not. With these exceptions, reduction in stimulation went along with reduction in viability. How­ ever, when the data were examined in another way, it be­ came apparent that there may be a dissociation between loss of viability and stimulatability. These data are pre­ sented in table III. Pairs of parameters which did not differ in the degree to which they decreased viability were com­ pared in their effect on stimulation. In the majority of instances, pairs of parameters which did not affect viability differently had a significant difference in effect on stimu­ lation.

Discussion This study has shown a marked effect of physico­ chemical alteration of the medium on response of normal

Table II. Effect of individual renal physicochemical parameterson viability of lymphocytes in presence of PHA1234 Parameter tested*

Viability, %2

Signifi­ cance3

concentration osmolality per liter mosm/kg Control* Sucrose

80 mM 150 m Ai 260 mM

310 375 450 600

52.81 32.18 28.65 9.80

±0.35 ±0.97 ± 1.26 ± 0.67

S S S S

183 mEq 228 mEq

375 450

34.65 ± l.ll 28.50 ± 1.60

S S

44 mEq 94 mEq

375 450

34.28 ± 1.45 20.17 ±0.83

S S

80 m M 160 mM 280 mM

375 450 600

40.77 ± 1.09 36.61 ± 0.85 36.80 ± 0.98

S S S

15 mEq

340

43.04 ± 1.60

S

Magnesium 1.5 mEq pH 6.5 6.8 8.0 8.4

312 -

52.26 _l 0.97 57.48 ± 1.25 55.40 ± 0.92 43.68 ± 1.01 37.92 ± 0.98

NS S

Sodium Potassium Urea

Calcium

1 on 2 3 4

-

s s s

5 replicates from S individuals, n = 25 for each except control based n = 200. After 72 h incubation. Mean ± SD of mean. Experimental vs. control by T test. S = 0.05 or better. Composition as described in table I.

Table III. Comparison of the effects on PHA stimulation of lymphocytes by pairs of parameters which had similar effects on viability

parameter

osmolality mosm/kg

viability. %2

parameter

osmolality mosm/kg

viability, %2

Control Sucrose Sucrose Sucrose Sucrose Sodium Sodium Sodium Potassium Potassium Urea Urea Urea pH pH

310 375 375 375 450 375 375 375 375 375 375 450 450 6.5 8.4

52.81 ±0.35 vs. 32.18 ± 0.97 vs. 32.18 ± 0.97 vs. 32.18 ±0.97 vs. 28.65 ± 1.26 vs. 34.65 ± 1.11 vs. 34.65 ± l.ll vs. 34.65 : l.ll vs. 34.28 ■ 1.45 vs. 34.28 • 1.45 vs. 40.77 ± 1.09 vs. 36.6! ± 0.85 vs. 36.61 ± 0.85 vs. 57.48 ± 1.25 vs. 43.68 :• 1.01 vs.

magnesium sodium sodium potassium sodium potassium urea urea urea urea calcium urea pH pH calcium

312 375 450 375 450 375 450 600 450 600 340 600 8.0 6.8 340

52.25 ± 0.97 34.65 ± l.ll 28.50 ± 1.60 34.28 ± 1.45 28.50 + 1.60 34.28 ± 1.45 36.61 ±0.85 36.80 ± 0.98 36.61 ± 0.85 36.80 0.98 43.04 ± 1.60 36.80 ± 0.98 37.92 0.98 55.40 ± 0.92 43.04 ± 1.60

* T test. 5 replicates from 5 individuals, n = 25 for each except control based on n = 200. 2 Mean ± SD of mean.

22,850 ± 14,017 ± 14,017 ± 14,017 ± 4.015 ± 13,494 ± 13,494 ± 13,494 ± 5.784 ± 5,784 ± 17,270 ± 14,334 ± 14.334 ± 2,956 ± 23.791 ±

1,208 vs. 854 vs. 854 vs. 854 vs. 350 vs. 951 vs. 951 vs. 951 vs. 989 vs. 989 vs. 1.596 vs. 1,199 vs. 1,199 vs. 705 vs. 1,774 vs.

Significance

20.224 ± 13,494 ± 2,762 ± 5,784 ± 2,762 ± 5.784 ± 14,334 ± 251 ± 14.334 ± 251 • 15,927 ± 251 ± 25,628 ± 14.094 ± 15,927 ±

1.619 951 229 989 229 989 1,199 36 1,199 36 1,244 36 2,420 1,460 1,244

NS NS