Clin. exp. Immunol. (1976) 23, 517-524.

In vitro quantitation of cell-mediated immunity in guinea-pigs by macrophage reduction of nitro-blue tetrazolium G. G. KRUEGER Division of Dermatology, University of Utah Medical Center, Salt Lake City, Utah B. E. OGD EN University of Utah College of Medicine, Salt Lake City, Utah. W. L. WESTON Division of Dermatology, University of Colorado School of Medicine, Denver, Colorado, U.S.A.

(Received 27 July 1975)

SUMMARY

In the cell-mediated immune (CMI) system lymphocytes from sensitized animals incubated with antigen manufacture and release lymphokines which activate the hexose-monophosphate shunt in macrophages. The rate-limiting enzyme of this activation is NADPH oxidase, the activity of which can be quantitated by the amount of nitro-blue tetrazolium reduced to formazan, a blue precipitate. Data is presented which demonstrates that lymphokine-activated macrophages can be microscopically quantitated, both in the direct and indirect assays, by counting the number of macrophages containing formazan precipitate. The indirect component of this assay correlates directly to the skin test diameter. Further, it correlates better to the skin test than another assay for CMI, the macrophage aggregation factor assay. The simplicity and reproducibility of this assay provides another method whereby lymphokine activation of physiological events in macrophages can be determined. INTRODUCTION In the cell-mediated immune (CMI) response, sensitized lymphocytes produce a number of biologically active products (lymphokines) in response to the sensitizing antigen. These soluble products are thought to be responsible for the amplification and recruitment seen in a CMI response (David, 1971). In recent years, methods have been described for the in vitro biological detection of these lymphokines; most are based upon changes effected in macrophage functions. Assay for the effect of these lymphokines on macrophage function such as the assay for migration inhibition factor (MIF) and macrophage aggregation factor (MAF) correlate well with delayed-type hypersensitivity skin tests and have received wide acceptance as tools for the study of CMI (Lolekha, Dray & Gotoff, 1970; Gotoff & Vizral, 1972; David, 1968). Nathan, Karnovsky & David (1971) demonstrated that macrophages incubated with fractions containing lymphokine exhibited increased phagocytosis, adherence and [l-14C]glucose oxidation. The increase in glucose oxidation noted in stimulated macrophages was attributed to oxidation through the hexose-monophosphate shunt (HMPS) pathway. The NADH or NADPH oxidases are felt to be the rate-limiting steps in the HMPS pathway (Rossi, Romeo & Patriarca, 1972), and as NADH and NADPH are oxidized in a system containing nitro-blue tetrazolium (NBT), NBT is reduced from a yellow soluble substance to a blue insoluble precipitate (formazan). Since lymphokine activated macrophages show Correspondence: Dr G. G. Krueger, Division of Dermatology, University of Utah Medical Center, Salt Lake City, Utah

84132, U.S.A.

517

518

G. G. Krueger, B. E. Ogden & W. L. Weston

increased glucose oxidation via the HMPS pathway, it was felt that this increase could be quantitated by the reduction of NBT. In this paper, we present data for an assay of CMI based upon the above phenomena and demonstrate a correlation between delayed-type hypersensitivity skin tests and NBT reduction by lymphokine-activated macrophages. MATERIALS AND METHODS Sensitization of animals. Each Hartley strain guinea-pig was immunized with 2-5 mg of heat-killed BCG (Research Foundation, Chicago, Illinois) in Freund's complete adjuvant (FCA). Intradermal tests were performed with 0-1 ml of second strength PPD (Connaught, Toronto, Canada), 14 days after immunization, and read at 24 hr for erythema and induration. Peritoneal exudate cells. A modification of the method described by Nathan, Remold & David (1971) was used. PEC were harvested 4 days after intraperitoneal injection of 15 ml of 1% case in, washed twice, suspended in RPMI-1640 with penicillin and streptomycin (P-S), counted, and viability determined by Trypan Blue exclusion (always > 90%). Preparation of lymphocyte supernatants. Lymphocyte suspensions prepared from the peripheral lymph nodes and spleen of each immunized guinea-pig were washed twice in HBSS, suspended in RPMI-1640-PS, counted, the viability determined (always > 70%), adjusted to a concentration of 1 x 107/ml, and divided into two aliquots. To one, 10 pg/ml preservative-free PPD (Parke Davis, Industrial Products Division, Detroit, Michigan) was added, the other served as a control; both were incubated at 370C, 5% CO2 and 95% humidity. At 24 and 48 hr, supernatants were collected, pooled, and an equivalent concentration of PPD was added to the controls. Supernatants were then filtered through a 0-45 gum millipore filter, divided into 3 ml aliquots and frozen at - 700C until used. NBT stain. Nitro-blue tetrazolium (Sigma Chemical Company, St Louis, Missouri) was dissolved in PBS (pH 7-4) to a final concentration of 0-2% with heating. The solution was cooled, sterile filtered, and stored in 5 ml aliquots at -20'C until use. Prior to each use, a mixture of RPMI-1640 and 5% dextrose (2: 1) with equal volumes of the 0-2% NBT in PBS was prepared and warmed to 370C. Direct NBT assay. To each chamber of an eight-chambered 1 x 3 in tissue culture slide (Lab-Tek, Miles Labs, Naperville, Illinois) 0 45 ml of a suspension of PEC (1 x 106/ml) from BCG-immunized guinea-pigs and 0 05 ml of either media alone or one of the test antigens was added to provide these final concentrations: PPD, 10 pg/ml; concanavalin A (Con A) (Calbiochem, San Diego, California), 10 pug/ml; and SK-SD (Varidase, Lederle Labs, Pearl River, New York, dialysed to remove preservative), 100 u/ml. Chambers were covered, incubated at 37°C, 5% CO2 and 95% humidity for 72 hr, at which time chambers were aspirated. Prepared NBT solution (0 3 ml) was added, and slides returned to the incubator for 30 mins. This solution was then aspirated from the chambers, chambers were removed, and slides were allowed to dry before fixing with methanol and counter-staining with safranin-O. The number of NBT-positive cells (those which contain formazan granules) per 100 counted at 1000 X was made twice per chamber, averaged and recorded. Results were evaluated as percentage of control [% NBT-positive Ag stimulated/% NBT-positive media alone x 100]. Indirect NBTassay. To each chamber 5 x 101 PEC from non-immunized animals in 0-2 ml RPMI-1640-PS, and 0 3 ml of either active or control supernatants was added. Cultures were then incubated, stained, and counted as described for the direct NBT assay. These supernatants were assayed with at least two different sources of PEC a total of five times. Results were evaluated as percentage of control. MAF assay. Direct and indirect macrophage aggregation assays, adapted from Lolekha et al. (1970) were performed simultaneously with direct and indirect NBT assays. Differences include casein-induced PEC, a final volume of 1-0 ml, and no foetal calf sera, in that unpublished data demonstrated no difference in aggregation with or without foetal calf sera. Protein adherence. Protein adherence was measured by a modification of the technique of Nathan et al. (1971). Following 72 hr incubation as above, each chamber was aspirated, the chamber units removed, slides rinsed, allowed to air dry, and chambers replaced with silicone cement. Adherent cellular material was solubilized with 1 N NaOH (18 hr), and protein determination was made by the method of Lowry et al. (1951).

RESULTS Direct NBT In chambers containing PEC from BCG-immunized guinea-pigs incubated with PPD, a higher number of NBT-positive cells was noted than in those chambers incubated with media alone (Fig. 1 and 2a). Using the paired t-test to compare each stimulated PEC

519

Quantitation of CMI by NBT

*-

'*~~"'

~O -

FIG. 1. (a) PPD-stimulated PEC from BCG-immunized animals. Reduced NBT, formazan, easily seen as blue-black material, at x 60. (b) Same as a, at x 600. (c) Same as a, without PPD at x 60. (d) Same as c, at x 600.

80 0>

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(b)

(a)

60

0 C

_

40

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C

2C

3C

S4C S5C S6C S7C

Animal number (stimulated (S) and control (C))

FK IC A2C A

2

A

3C A4 C A5C A 6C

3

4

5

A C

6

7

Animal number (active (A) and control (C))

FIG. 2. (a) Comparison of NBT-positive PEC from BCG-immunized animals in PPD-stimulated controls, the direct assay. Experiments done in quadruplicate, each point represents actual number of NBT-positive cells/100 counted, lines connect each stimulated value with its control. (b) Comparison of NBT-positive PEC from non-immunized animals incubated with active (lymphokine containing) and control supernatants, the indirect assay. Five indirect assays performed with lymphokine from each animal; PEC from at least two animals. Each point represents the percentage of NBT-positive cells/100 counted for each culture; lines connect each stimulated value with its control. versus

culture to its control, differences observed between PPD and control cultures in all seven animals were highly significant (P 130%) and as a group was not significant when compared to control. We did not skin test these animals to SK-SD and thus did not exclude the possibility that any of these animals might have been previously exposed to streptococcal antigen. For another control of the direct assay, PPD, SK-SD and Con A were incubated with PEC from non-immunized guinea-pigs. The differences between the PPD- and SK-SD-

520

G. G. Krueger, B. E. Ogden & W. L. Weston TABLE 1. Specificity of direct assay

BCG-immunized

Mean Non-immunized

Animal

PPD*

Con-A*

SK-SD*

1 2 3 4 5 6 7

509± 27 536± 116 435±64 400±18

559± 178 326±49 271 ±26 180±24 208±31 246±44 131±9 281± 58 104±12 88+±05 96±8

117±18 111±22 159± 13 114±6

1 2

Mean

193±13 255±33

178±13 358± 59 112±12 114±12 113+1 0

106±14 128±22 84±5 121±10 111+6 107±8 109+2

* (No. of NBT-positive cells Ag-stimulated cultures/no. of NBT-positive cells in control cultures) x 100 = percentage of control. Values = mean of four replicate cultures ± s.e.m.

TABLE 2. Direct MAF versus NBT

Animal

PPD skin test*

1 2 3 4 5 6 7

17 13 14 13 20 14 11

Efficacy:

MAFt

NBTJ

PercentageS

0/4 4/4 509+ 27 0/4 4/4 536± 116 4/4 435±64 4/4 2/4 4/4 400±80 4/4 4/4 193±13 4/4 4/4 255±33 2/4 4/4 178+13 16/28 = 57% 28/28 = 100% Mean: 358±57

P value¶

< 0-01