International Immunology

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International Immunology, Vol. 11, No. 11, 1811-1818, November 1999
© 1999 Japanese Society for Immunology

Development of an assay to measure in vivo cytokine production in the mouse

Fred D. Finkelman and Suzanne C. Morris¹

Division of Immunology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
¹ Cincinnati Veterans Administration Medical Center, Cincinnati, OH 45220, USA

Correspondence to: F. Finkelman, Division of Immunology, University of Cincinnati College of Medicine, PO Box 670563, Cincinnati, OH 45267-0563, USA

	Abstract

Top Abstract Introduction Methods Results Discussion Note added in proof References

 
The short in vivo lifespan of many cytokines can make measurement of in vivo cytokine production difficult. A method was developed to measure in vivo IL-4 and IFN- production that eliminates this problem. Mice are injected with a biotin-labeled neutralizing IgG anti-IL-4 or anti-IFN- mAb and bled 2–24 h later. Secreted cytokine is captured by the biotin-labeled mAb to produce a complex that has a relatively long in vivo half-life and consequently accumulates in serum. Serum concentrations of the complex are determined by ELISA, using wells coated with an antibody to a second epitope on the same cytokine to capture the complex. This technique is specific and increases sensitivity of detection of secreted IL-4 at least 1000-fold. The amount of cytokine measured is directly proportional to the amount produced and relatively independent of the site of cytokine production. Furthermore, because mice are injected with small quantities of biotin-labeled anti-cytokine mAb, which sample, rather than neutralize, all secreted cytokines, cytokine-dependent responses are not inhibited. The in vivo half-lives of the cytokine–anti-cytokine mAb complexes are sufficiently short to allow cytokine production to be measured every 2–3 days in the same mice. Thus, use of this assay provides a practical and relatively simple and inexpensive way to measure ongoing in vivo cytokine production. Furthermore, the techniques that have been developed to measure in vivo production of IL-4 and IFN- can be applied to in vivo measurement of other molecules that have a short in vivo lifespan, including other cytokines.

Keywords: blood, cytokines, in vivo animal models, rodent, T_h1, T_h2

	Introduction

Top Abstract Introduction Methods Results Discussion Note added in proof References

 
Because the nature of an immune response is controlled, to a considerable extent, by cytokines produced during the response (1), characterization of cytokine production is important for understanding the nature of the response. Several techniques have been developed to determine cytokine production, which measure cytokine mRNA expression or cytokine protein synthesis or secretion (2–11). Limitations of these techniques include the possibilities that cytokine mRNA levels may not correlate well with levels of cytokine protein production and that cellular cytokine protein content may not correlate well with cytokine secretion. In addition, because ex vivo cytokine production by cells from immunized animals is often below detectable levels, it is frequently necessary to re-stimulate immune cells in vitro with antigen or mitogen to reveal their capacity to produce cytokine (4,7,8,11). These techniques work well to increase cytokine production to detectable levels; however, it is not known whether cytokine production by cells re-stimulated in vitro always correlates well with cytokine production in vivo. Furthermore, methods that measure cytokine mRNA or depend on in vitro re-stimulation to measure cytokine protein generally require the sacrifice of a group of animals at each time point measured, as well as the sacrifice of an additional group if it is desired to correlate cytokine responses with another biological response. Consequently, large numbers of experimental animals frequently must be used in such studies.

It would be preferable, in many circumstances, if measurement of serum cytokine levels could be used to determine cytokine production. Unfortunately, many cytokines, such as IL-4, are produced in such small amounts and are so rapidly excreted, catabolized or utilized that the quantities that accumulate in serum are undetectable by available assay systems (12).

The studies described here were designed to develop and test a method that had the advantages of measuring serum cytokine levels, but would increase the in vivo lifespan of cytokines by inhibiting their utilization, catabolism and renal excretion. Because previous studies have demonstrated that preformed complexes of cytokines with neutralizing anti-cytokine mAb can have a considerably longer in vivo lifespan than the free cytokines (12), we examined whether the injection of mice with a small quantity of a neutralizing anti-cytokine mAb could `capture' the cytokine and cause it to accumulate in serum. This might cause serum cytokine levels to increase in proportion to their production, so that relative rates of in vivo cytokine production could be determined by measuring serum concentrations of cytokine–anti-cytokine mAb complexes by ELISA. Results of our studies demonstrate that this in vivo assay, which we have named the Cincinnati cytokine capture assay (CCCA), provides an accurate and efficient way to measure relative in vivo IL-4 production and that a modification of the same assay can measure in vivo production of IFN-.

	Methods

Top Abstract Introduction Methods Results Discussion Note added in proof References

 
Mice
BALB/c and C57BL/6 female mice and athymic nude male mice were purchased from the NCI (Bethesda, MD), and used at 8–16 weeks of age. C57BL/6.IL-4-deficient mice (13) were originally obtained form Dr Herbert Morse (NIH, Bethesda, MD) and C57BL/6.IFN--deficient mice (14) were originally purchased from Jackson (Bar Harbor, ME). C57BL/6.IL-4-deficient and C57BL/6.IFN--deficient mice were bred to each other to produce mice doubly deficient in IL-4 and IFN- on a C57BL/6 background. Offspring of these matings were genotyped by PCR for IL-4 and IFN- (15). The IL-4 PCR used 5'-GCACAGAGCTATTGATGGGTC-3', 5'-GCTGTGAGGACGTTTGGC-3' and 5'-TCAGGACATAGCGTTGGC-3' as primers. The wild-type IL-4 allele yielded a 444 bp product, while the neo-disrupted allele yielded a 576 bp product with a faint band at 1577 bp. The IFN- PCR used 5'-AGAACTAAGTGGAAGGGCCCAGAAG-3', 5'-AGGGAAACTGGGAGAGGAGAAATAT-3' and 5'-TCAGCGCAGGGGCGCCCGGTTCTTT-3' as primers. The wild-type IL-4 allele yielded a 220 bp product, while the neo-disrupted allele yielded a 1150 bp product. Mice that selectively express an IL-4 transgene in their lungs and background controls (16) were a gift of Dr Jeffery Whitsett (Children's Hospital Research Foundation, Cincinnati, OH).

Antibodies and immune reagents
Goat anti-mouse IgD antibody (GaM) and normal goat IgG were produced as described (17). Hybridomas that produce hamster anti-mouse TCR mAb (145-2C11) (18) or neutralizing rat IgG anti-mouse IFN- (R4-6A2) (19) were obtained from the ATCC (Rockville, MD). A hybridoma that secretes an IgG2b mAb of the b allotype that binds to mouse IgD of the a allotype (H^a/1) (20) and a hybridoma that secretes an IgG2a mAb of the b allotype that binds to mouse IgD of the a allotype (FF1-4D5) (21) were grown as ascites in Pristane-primed C57BL/6 mice. Hybridomas that produce rat IgG neutralizing mAb to IL-4 (BVD4-1D11) (22), IL-5 (TRFK-5) (23) or IL-3 (MP2-8F8) (22), or non-neutralizing mAb to IL-4 (BVD6-24G2.3) (22) or IL-3 (MP2-43D11) (24) were obtained from the ATCC with the permission of DNAX (Palo Alto, CA) and the assistance of Dr Robert Coffman. A hybridoma that produces a non-neutralizing rat IgG anti-IFN- mAb (AN-18) (25) was a gift of Dr Anne O'Garra (DNAX). All of these hybridomas were grown as ascites in Pristane-primed athymic nude mice. All mAb were purified from ascites by ammonium sulfate precipitation and ion-exchange chromatography as described, using isotype-specific anti-rat Ig antisera (ICN, Aurora, OH) and gel double-diffusion analysis to identify antibody-rich fractions. AECM-Ficoll was purchased from Biosearch (San Raphael, CA). Alkaline phosphatase conjugated to streptavidin was purchased from Jackson ImmunoResearch (West Grove, PA). Purified, recombinant mouse IL-4 was a generous gift of the Schering-Plough Research Institute (Kenilworth, NJ), recombinant mouse IL-3 and IFN- were generous gifts of PharMingen (La Jolla, CA), and purified, recombinant mouse IL-12 was a generous gift of Hoffman LaRoche (Piscataway, NJ). AECM-Ficoll and some mAb were biotin-conjugated, using biotin N-hydroxy-succinimide (Calbiochem-Behring, La Jolla, CA), as described (26), at a 1:10 (w/w) biotin:protein ratio.

ELISA
Immulon II 96-well microtiter plates (Dynatech, Chantilly, VA) were incubated at room temperature for 2 h with 10 µg/ml of the appropriate non-neutralizing anti-cytokine mAb in 0.1 M Tris, pH 8.3. Plates were then washed 10 times with deionized H₂O, using a Microwash II microtiter plate washer (Skatron, Sterling, VA) and blocked overnight at 4°C using 5% Carnation non-fat dry milk powder and 0.2% NaN₃ in 0.1 M Tris, pH 8.3 (5% milk buffer). To titer sera for a cytokine–anti-cytokine mAb complex, 25 µl of serial 4-fold dilutions of sera (starting with undiluted sera or sera diluted 1:2, 1:4 or 1:10 with 0.1M Tris, pH 8.3, supplemented with 0.25% Carnation non-fat dry milk powder, 0.2% NaN₃ and 0.05% Tween 20) (MBTA) was added in duplicate to the microtiter plate wells. After 30 min, all wells were filled with MBTA, using a Nunc Immunowash 12 hand washer (Nunc, Kamstrup, Denmark), aspirated and filled with MBTA, then washed 10 times with deionized H₂O in the microtiter plate washer. Plates were then filled with MBTA and incubated for 5 min at room temperature, and again washed with deionized water. Similar MBTA/deionized water wash steps were performed after every subsequent incubation with a reagent. Alkaline phosphatase–streptavidin (25 µl diluted 1:2000 in MBTA) was then added to wells, plates were incubated for 1 h at room temperature and again washed. To increase ELISA sensitivity, incubation of wells with alkaline phosphatase–streptavidin was followed by incubation with 25 µl of biotin–AECM-Ficoll (1 µg/ml in MBTA), washing and a second incubation with alkaline phosphatase–streptavidin. Following the final incubation with alkaline phosphatase–streptavidin, plates were washed and wells were filled with 200 µl of an alkaline phosphatase substrate [p-nitrophenylphosphate (Calbiochem-Behring), 1 mg/ml, in 0.1 M Tris and 0.01% 3 M MgCl_2, pH 9.8] and incubated for 0.5–24 h, after which the A₄₀₅ of each well was read with a Multiskan MS ELISA plate reader (Labsystems, Helsinki, Finland). Complexes of recombinant cytokine and biotin-labeled anti-cytokine mAb were used as standards. Our IL-4 standard contained 100 pg/ml of recombinant mouse IL-4 and 15 ng/ml of biotin–BVD4-1D11; our IFN- standard contained 1 ng/ml of recombinant mouse IFN- and 150 ng/ml of biotin–R46A2. Then 25 µl of the appropriate standard was added to microtiter plate wells and five serial 4-fold dilutions of the standard were performed. Plates were further treated as described for sera. Titers were determined as the serum dilution or cytokine concentration that would elicit a given A₄₀₅ after a prescribed incubation period. Serum cytokine concentrations were determined by comparing serum titers to titers obtained for a known cytokine concentration.

Since the experiments described in this paper were performed, five changes have been made in our ELISA protocol that lower background and decrease the time required for the assay without decreasing its sensitivity. (i) Milk buffer is centrifuged for 10 min at 1000 r.p.m. to remove insoluble material. (ii) Azide is omitted from all buffers, which are now prepared weekly and stored at 4°C. (iii) ELISA plates are incubated for 1 h at 37°C, instead of 30 min at room temperature, after adding serum dilutions. (iv) The incubation with biotin–AECM-ficoll and the second incubation with streptavidin–alkaline phosphatase are no longer performed. (v) The starting concentration of the IL-4 standard is now 1 ng/ml instead of 100 pg/ml.

Serum IgE levels were also determined by ELISA, as previously described (27).

Assay strategy and description
The CCCA is based on the idea that an injected, neutralizing anti-cytokine mAb will complex with a secreted cytokine and cause it to accumulate in blood as a cytokine–anti-cytokine mAb complex by protecting it from utilization by preventing binding to cellular cytokine receptor, from renal excretion and from catabolism. If the injected anti-cytokine mAb is biotin-labeled, the serum concentration of the cytokine–anti-cytokine mAb complex can be determined by ELISA, using microtiter plate wells coated with a second antibody that binds to a different epitope on the same cytokine molecule to capture the biotin–anti-cytokine mAb–cytokine complex and detecting the bound complex with enzyme linked to streptavidin, followed by addition of a chromogenic substrate for the enzyme and determination of the concentration of the colored product with a microtiter plate ELISA reader that is set for the appropriate wavelength. This process is illustrated in Fig. 1.

View larger version (17K): [in this window] [in a new window]   Fig. 1. CCCA strategy. Mice are injected i.v. with a biotin-labeled neutralizing anti-cytokine antibody, which binds secreted cytokine and prevents its excretion, utilization or degradation. The biotin–anti-cytokine antibody–cytokine complex is detected by ELISA, using an antibody to a second epitope on the same cytokine molecule to bind the biotin–anti-cytokine antibody–cytokine complex and enzyme linked to streptavidin, followed by a chromogenic substrate for that enzyme, to detect the bound complex.

 

	Results

Top Abstract Introduction Methods Results Discussion Note added in proof References

 
The CCCA increases the sensitivity of measurement of in vivo IL-4 production
To stimulate in vivo IL-4 production, BALB/c mice were injected i.v. with saline or with 800 µg of affinity-purified GaM, which stimulates peak IL-4 production 5–6 days after antibody injection (2). To determine if in vivo capture of secreted IL-4 by a biotin-labeled anti-IL-4 mAb would make it easier to detect IL-4 in serum, mice injected 5 days earlier with saline or with GaM were injected i.v. with 10 µg of biotin–BVD4-1D11 (anti-IL-4) and bled 1 day later. As a control, GaM-injected mice were bled 1 day after they had received biotin-labeled anti-IL-5 mAb (TRFK-5) or no additional treatment. Serum levels of IL-4 were determined by ELISA, using microtiter plates coated with the non-neutralizing anti-IL-4 mAb, BVD6-24G2.3, as described above, with the exception that an extra step was performed: biotin–BVD4-1D11 was added to all wells after addition of sera to detect bound IL-4 because only some mice had received this mAb in vivo. Results of this study (Fig. 2) demonstrate that ~25 pg/ml of IL-4 was detected in unimmunized mice that received biotin–anti-IL-4 mAb, while GaM treatment caused IL-4 captured by biotin–anti-IL-4 to increase ~100-fold. No IL-4 (<7 pg/ml) was detectable in unimmunized or GaM-immunized mice that had received no biotin-labeled mAb or biotin–anti-IL-5. Thus, the in vivo capture of IL-4 with biotin–anti-IL-4 mAb increases the ability to detect in vivo IL-4 production by >300-fold.

View larger version (15K): [in this window] [in a new window]   Fig. 2. The CCCA allows detection of IL-4 secreted in vivo. BALB/c mice (five per group) were injected i.v with saline or with 800 µg of affinity-purified GaM on day 0 and with saline, 10 µg of biotin–TRFK-5 (anti-IL-5 mAb) or 10 µg of biotin–BVD4-1D11.2 (anti-IL-4 mAb) on day 4. Mice were bled on day 5. Serum levels of IL-4 were determined by ELISA. Microtiter plate wells were coated with BVD6-24G2.3 (anti-IL-4 mAb that binds an epitope separate from that bound by BVD4-1D11.2) and blocked. Serial dilutions of serum or a recombinant mouse IL-4/biotin–BVD4-1D11.2 standard, biotin–BVD4-1D11.2, alkaline phosphatase–streptavidin and substrate were sequentially added to wells. Plates were read at A405 the next day. IL-4 concentrations were calculated as described in Methods. Geometric means + SE are shown in this figure and in subsequent figures.

 
Cytokine detection by the CCCA is specific
To determine the specificity of the CCCA for IL-4, we measured serum IL-4 levels, as stimulated by anti-CD3 mAb treatment (28), in C57BL/6 wild-type and IL-4-deficient mice (Fig. 3). Serum IL-4 levels in conventional, unimmunized mice that received 10 µg of biotin–BVD4-1D11.2 and were bled 2 h later were ~7 pg/ml. Serum IL-4 levels increased >4000-fold if conventional mice were injected i.v. with 10 µg of anti-CD3 mAb 2 h before i.v. injection of 10 µg of biotin–anti-IL-4 mAb and were bled 2 h after the second injection. In contrast, no IL-4 (<3 pg/ml) was detectable in the serum of anti-CD3 mAb-immunized, biotin–anti-IL-4-treated, IL-4-deficient mice.

View larger version (11K): [in this window] [in a new window]   Fig. 3. The IL-4 CCCA is specific for IL-4. C57BL/6 wild-type mice were injected i.v. with 10 µg of biotin–BVD4-1D11.2 and saline or 10 µg of 2C11 (anti-CD3 mAb). C57BL/6.IL-4-deficient mice were injected with biotin–BVD4-1D11.2 and anti-CD3 mAb. All mice (two to three per group) were bled 4 h later. Serum IL-4 levels were assayed by CCCA as in the legend to Fig. 1. Recombinant mouse IL-4/biotin–BVD4-1D11.2 complex was used as a standard in this figure and in subsequent figures.

 
In vivo production of IL-4 is optimally detected by injection of 10 µg of biotin–anti-IL-4 mAb
Our initial experiments arbitrarily injected mice with a 10 µg dose of anti-IL-4 mAb to capture secreted IL-4. To determine if a higher or lower dose of anti-IL-4 mAb would improve results, BALB/c mice were injected with 800 µg of GaM to stimulate IL-4 production and, 5 days later, with 0.4–50 µg of biotin–anti-IL-4 mAb to capture secreted IL-4. Mice were bled 5 h after biotin–anti-IL-4 mAb injection and serum IL-4 levels were determined by ELISA (Fig. 4). Serum IL-4 levels were nearly as high in mice injected with 10 µg of biotin–anti-IL-4 as in mice injected with 50 µg of biotin–anti-IL-4 mAb, but were significantly lower in mice injected with 2 µg of biotin–anti-IL-4 mAb. Because of this result, a 10 µg dose of biotin–anti-IL-4 mAb was used in all subsequent experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Biotin–anti-IL-4 mAb at 10 µg is sufficient to capture secreted IL-4. BALB/c mice (three per group) were injected i.v. with 800 µg of affinity-purified goat anti-mouse IgD antibody. Five days later mice were injected i.v. with 0.4–50 µg of biotin–BVD4-1D11.2. Mice were bled 5 h after that and serum IL-4 levels were determined as in the legend to Fig. 1.

 
CCCA detection of IL-4 is proportional to the quantity of IL-4 injected into mice and is largely independent of the site of IL-4 injection
To determine if the concentration of serum IL-4 detected by the CCCA is proportional to the amount of IL-4 secreted and if the site of IL-4 secretion influences the quantity of IL-4 detected, unimmunized mice were injected i.v. with 10 µg of biotin–anti-IL-4 mAb and, 1 h later, i.v., i.p. or s.c. with 1000, 250 or 63 ng of recombinant IL-4. Mice were bled 4 h after IL-4 injection and serum IL-4 levels were determined by ELISA (Fig. 5). Results demonstrated that serum IL-4 levels were proportional to the quantity of IL-4 injected and were not influenced to any appreciable extent by the route of IL-4 injection. These observations suggest that the CCCA can be used to measure IL-4 production and that IL-4 produced at the three different sites tested should be detectable. To further examine this last point, we investigated whether the CCCA could detect increased IL-4 production in mice in which a transgene that induces increased IL-4 production is regulated by a lung-specific, Clara cell-derived, promoter that causes IL-4 to only be produced in the lungs (IL-4 LTgn mice) (16). Serum IL-4 levels, as detected by the CCCA, were ~100-fold greater in biotin–anti-IL-4 mAb-treated IL-4 LTgn mice than in similarly treated wild-type mice and were not detectable in untreated IL-4 LTgn mice (Fig. 6). Thus, the CCCA can detect IL-4 that is produced at different sites of the body.

View larger version (14K): [in this window] [in a new window]   Fig. 5. The CCCA equally detects IL-4 in different sites. BALB/c mice (five per /group) were injected i.v. with 10 µg of biotin–BVD4-1D11.2 and 1 h later, were injected i.p., i.v. or s.c. with 62.5, 250 or 1000 ng of IL-4. Mice were bled 5 h later and serum IL-4 levels were determined as described in the legend to Fig. 1.

 

View larger version (13K): [in this window] [in a new window]   Fig. 6. The CCCA can detect IL-4 secreted in the lungs. Wild-type and IL-4 LTgn mice (three per group) were injected i.v. with saline or with 10 µg of biotin–anti-IL-4 mAb and bled 1 day later. Serum IL-4 levels were determined as in the legend to Fig. 1.

 
The CCCA can be used to detect IL-4 secretion at multiple timepoints in the same mouse and does not affect IL-4-dependent biological responses
Because preformed IL-4–anti-IL-4 mAb complexes have an in vivo half-life of ~1 day (12), we expected that it might be possible to repeatedly determine IL-4 production in the same mice, provided that sufficient time was allowed for loss of the initial bolus of injected biotin–anti-IL-4 mAb. To test this possibility, groups of BALB/c mice were immunized i.v. with 100 µg each of two allo-anti-mouse IgD mAb (H^a/1 and FF1-4D5) and injected with biotin–anti-IL-4 mAb 2 and 4 days after immunization or 1, 3 and 5 days after immunization. All mice that received biotin–anti-IL-4 mAb were bled 1 day after each injection of biotin–anti-IL-4 mAb and sera were analyzed for IL-4 content by ELISA. These mice and an additional group of anti-IgD mAb-immunized mice that was never treated with anti-IL-4 mAb were bled 8 days after anti-IgD mAb injection and serum IgE levels were determined by ELISA. Results of this experiment (Fig. 7) demonstrate that serum IL-4 levels slowly increased, starting 2 days after anti-IgD mAb injection, peaked at day 5 and started to decrease by day 6. Similar IgE levels were observed in all groups of mice. These results demonstrate that the same mice can be injected more than once to determine IL-4 production at different timepoints and that even multiple injections of biotin–anti-IL-4 mAb do not influence the magnitude of an IL-4-dependent response.

View larger version (19K): [in this window] [in a new window]   Fig. 7. The CCCA can be used to detect the kinetics of in vivo IL-4 production by repeated sampling of the same mice. BALB/c mice (five per group) were injected i.v. with 100 µg each of Ha/1 and FF1-4D5 anti-IgD mAb on day 0. Mice either received no additional treatment or were injected i.v. with 10 µg of biotin–BVD4-1D11.2 on days 2 and 4, and were bled on days 3 and 5 or were injected i.v. with 10 µg of biotin–BVD4-1D11.2 on days 1, 3 and 5, and were bled on days 2, 4 and 6. All mice were bled on day 8. IL-4 ELISAs were performed on sera obtained on days 2–6. Mean ELISA (serum dilution versus A405) results for each bleed are shown as a line graph. Means + SE for day 8 total serum IgE levels were determined by ELISA and are shown as a bar graph.

 
The CCCA can be used to specifically measure IFN- production
To determine if the CCCA technique could be used to measure cytokines besides IL-4, we used a similar procedure to the IL-4 CCCA to measure in vivo production of IFN-. Anti-CD3 mAb-treated BALB/c mice were injected with 10–160 µg of biotin–anti-IFN- mAb (R46A2) at the time of anti-CD3 mAb treatment and bled 4 h later. Results showed near maximal IFN- detection at a 40 µg dose but considerably less IFN- detection at a 10 µg dose (Fig. 8, upper panel). Doses of 50 µg of biotin–anti-IFN- mAb were injected in subsequent experiments. At this dose, serum IFN- was detectable in unimmunized BALB/c mice and increased ~1000-fold in anti-CD3 mAb-immunized mice (Fig. 8, middle panel). No IFN- was detected in the serum of anti-CD3 mAb-immunized C57BL/6.IFN--deficient mice (Fig. 8, lower panel).

View larger version (19K): [in this window] [in a new window]   Fig. 8. The CCCA detects secreted IFN- sensitively and specifically. (Upper panel) BALB/c mice (three per group) were injected i.v. with 10 µg of anti-CD3 mAb (2C11) plus 10, 40 or 160 µg of biotin–anti-IFN- mAb (R46A2) and bled 4 h later. Serum IFN- levels were determined by ELISA, using microtiter plates coated with a mAb (AN-18) that binds to a different IFN- epitope than that bound by R46A2. (Middle panel) BALB/c mice (three per group) were injected i.v. with 50 µg of biotin–anti-IFN- mAb or with 50 µg of biotin–anti-IFN- mAb plus 10 µg of anti-CD3 mAb. Mice were bled 4 h later and serum IFN- levels were determined by ELISA. (Lower panel) C57BL/6 wild-type and IFN--deficient mice (three per group) were injected i.v. with 10 µg of anti-CD3 mAb plus 50 µg of biotin–anti-IFN- mAb and were bled 4 h later. Serum IFN- levels were determined by ELISA.

 
The CCCA can simultaneously detect production of more than one cytokine in the same mouse
An assay that could be used to simultaneously detect the production of more than one cytokine in the same mouse would be more efficient and would use fewer animals than one that could only detect a single cytokine in one animal. To determine whether the CCCA could be used to simultaneously detect production of three cytokines, C57BL/6 wild-type, IL-4-deficient, IFN--deficient and IL-4/IFN--double-deficient mice were simultaneously injected i.v. with 10 µg of anti-CD3 mAb, 10 µg of biotin–anti-IL-4 mAb, 50 µg of biotin–anti-IFN- mAb and 25 µg of biotin–anti-IL-3 mAb, and bled 4 h later. Results of this experiment (Fig. 9) demonstrated that all three cytokines were detected in wild-type mice, and that IL-3 and IL-4, but not IFN-, were detected in IFN--deficient mice. IL-3, IFN- and small but measurable quantities of IL-4 (<1/100 the amount detected in wild-type or IFN--deficient mice) appeared to be detected in IL-4-deficient mice, and IL-3 and similar small quantities of IL-4, but no IFN-, were detectable in IL-4/IFN--double-deficient mice. These results indicate that the CCCA can be used to measure the production of more than one cytokine at the same time, with the stipulation that attempted measurement of multiple cytokines increases the background for detection of IL-4, so that small amounts of IL-4 appear to be made even when IL-4 production is truly absent.

View larger version (22K): [in this window] [in a new window]   Fig. 9. The CCCA can be used to simultaneously detect secretion of IL-3, IL-4 and IFN- in the same mice. C57BL/6 wild-type, IL-4-deficient, IFN--deficient and IL-4/IFN--double-deficient mice (three per group) were injected i.v. with 10 µg of anti-CD3 mAb plus 10 µg of biotin–anti-IL-4 mAb, 25 µg of biotin–anti-IL-3 mAb and 50 µg of biotin–anti-IFN- mAb, and bled 4 h later. Serum IL-4 and IFN- levels were determined by ELISA as described above; serum IL-3 levels were determined by ELISA using microtiter plates coated with an anti-IL-3 mAb (MP2-43D11) that binds an IL-3 epitope different from that bound by MP2-8F8.

 
IL-12 enhances IFN- production and suppresses IL-4 production in GaM-treated mice
To determine if the CCCA could be used to detect a shift in cytokine production during an immune response, we used it to examine the effects of IL-12 on GaM-treated mice. Previous studies, which used RT-PCR to characterize in vivo cytokine gene expression, demonstrated that GaM induces a larger percentage increase in IL-4 than IFN- mRNA expression and that the relative production of these two cytokines is reversed if mice are simultaneously treated with IL-12 (29). CCCA detected a 9-fold increase in serum IL-4 concentration and a 3-fold increase in serum IFN- concentration 6 days after immunization of BALB/c mice with 400 µg of GaM plus 400 µg of normal goat IgG. The magnitude of the increase in IL-4 production was probably greater than was revealed by the assay, because simultaneous measurement of IL-4 and IFN- increases background IL-4 determinations, as noted above. Consistent with previous RT-PCR data (29), daily treatment of GaM-immunized mice with 500 ng/day of IL-12, starting on the day of GaM-injection reversed the cytokine nature of the response by decreasing serum IL-4 levels by a factor of 3.6 and causing a 152-fold increase in serum IFN- levels (Fig. 10). Thus, the CCCA can be used to `T_h-type' cytokine responses and detect the in vivo effects of immunomodulators on cytokine production.

View larger version (21K): [in this window] [in a new window]   Fig. 10. IL-12 inhibits GaM-induced IL-4 production and enhanced GaM-induced IFN- production, as measured by the CCCA. BALB/c mice (five per group) were left untreated or were injected i.v. with 400 µg of GaM plus 400 µg of normal goat IgG. GaM-treated mice received daily i.p. injections of saline or 500 ng of IL-12. Six days after GaM injection mice were injected i.v. with 10 µg of biotin–anti-IL-4 mAb and 50 µg of biotin–anti-IFN- mAb, and were bled 2 h later. Serum IL-4 and IFN- levels were determined by ELISA.

 

	Discussion

Top Abstract Introduction Methods Results Discussion Note added in proof References

 
Our observations demonstrate that the CCCA is a simple, objective, sensitive and specific technique for the measurement of in vivo cytokine secretion. Use of this technique avoids the potential problems of measuring cytokine mRNA as a substitute for cytokine protein, measuring intracellular cytokine content as a substitute for cytokine secretion or measuring cytokine secretion after ex vivo re-stimulation as a substitute for in vivo secretion. In addition, the ability to repeatedly measure cytokine secretion in the same mice, without interfering with an ongoing cytokine-dependent immune response, can, in some experiments, considerably decrease experimental animal requirements and costs.

The CCCA does, however, have limitations. First, it does not reveal the identity of cytokine-secreting cells. Secondly, foreign anti-cytokine mAb can elicit antibody responses that prevent their capture of secreted cytokine. This can cause false negative results in some studies if mice are repeatedly injected with the mAb for >10 days (data not shown). Thirdly, some anti-cytokine mAb react with each other in ways that interfere with the simultaneous measurement of multiple cytokines. For example, biotin-labeled anti-IFN- mAb (R46A2) interacts with the anti-IL-4 mAb (BVD6-24G2.3) that is used to coat ELISA plates to capture IL-4-containing complexes (data not shown). This interaction probably would not cause problems in measuring large increases in IL-4 production, but does cause problems in detecting small increases and problems when it is necessary to accurately determine baseline IL-4 concentrations. Under such circumstances, it is preferable to use the CCCA to measure the production of a single cytokine in a single animal, because no false positive results have been observed under those conditions.

A fourth limitation of the CCCA is that precise quantitation of cytokine secretion may not be possible. Although one strength of the CCCA is the direct relationship between quantity of cytokine secreted (or injected) and quantity detected, there is not necessarily a 1:1 proportionality between these two parameters. As the quantity of cytokine secreted increases, the percent that is bound by anti-cytokine antibody may decrease, because of a decreasing ratio of secreted cytokine to injected anti-cytokine mAb, or increase, because of increasing saturation of cell membrane and secreted cytokine receptors that compete for cytokine with injected anti-cytokine mAb. Consequently, a 10-fold increase in the quantity of cytokine detected by the CCCA might not precisely represent a 10-fold increase in cytokine secretion. Nevertheless, the several log increases in cytokine production that have been detected by the CCCA in some experiments indicate that this technique can be used, at least qualitatively, to detect changes in cytokine production.

A fifth limitation of the CCCA is that it would probably be less practical to use in larger animals than in mice. Assuming that the quantity of biotin–anti-cytokine mAb needed to detect cytokine production is proportional to an animal's extracellular fluid volume, the expense of performing the CCCA would be considerably greater in large animals than in mice. The requirement to inject foreign mAb would make the CCCA particularly difficult to use in humans, unless anti-cytokine antibodies or soluble cytokine receptors were already being administered for a different diagnostic or a therapeutic purpose.

Despite all of these limitations, the ease of use of the CCCA and its ability to repeatedly measure whole body in vivo cytokine secretion should make it a valuable tool for small animal studies, particularly as CCCA for additional cytokines are developed. Our recent, surprising observations that Stat6-deficient mice can make normal type 2 cytokine responses to some antigens (30,31) provides an example of the usefulness of the CCCA in a setting in which different results have been obtained from standard in vitro assays of cytokine production (32–34). The usefulness of the CCCA in obtaining these results suggests that this assay may have general utility in the detection and quantitation of other secreted molecules that have a short in vivo lifespan, including cytokines other than IL-4 and IFN-, and some polypeptide hormones and secreted tumor antigens.

	Note added in proof

Top Abstract Introduction Methods Results Discussion Note added in proof References

 
Subsequent to the acceptance of this paper for publication, we have discovered that the in vivo half-life of IFN-—biotin-anti-IFN- mAb complexes is much longer than the in vivo half-life of IL-4—biotin-anti-IL-4 mAb complexes. Consequently, while it is possible to repeatedly measure IL-4 production multiple times in the same mouse, IFN- production can only reliably be measured once per mouse. In addition, the IFN- CCCA, unlike the IL-4 CCCA, becomes considerably more sensitive if mice are bled 24 hours, rather than 2 hours, after injection of biotin-labeled mAb.

	Acknowledgments

 
Work described here was supported by the US Department of Veterans Affairs, and NIH grants RO1-AI35987 and RO1-AI37180. We are grateful to Dr Robert Coffman for his gift of hybridomas, Genetics Institute for its gift of recombinant mouse IL-12, Schering-Plough Research Institute for its gift of IL-4, PharMingen for its gift of IL-3 and IFN-, Dr Herbert Morse for his gift of C57BL/6.IL-4-deficient mice, and Dr Anne-Sophie Gadenne, Ryan Swisher, Tatyana Orekhova and Edward Roach for their excellent technical assistance.

	Abbreviations

 

CCCA Cincinnati cytokine capture assay

GaM affinity-purified goat antibody specific for mouse IgD

MBTA 0.1 M Tris, pH 8.3, supplemented with 0.25% Carnation non-fat dry milk powder, 0.2% NaN3 and 0.05% Tween 20

	Notes

 
Transmitting editor: R. Coffman

Received 20 May 1999, accepted 25 July 1999.

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Top Abstract Introduction Methods Results Discussion Note added in proof References

 

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