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Cytokine MAPPing in autoimmunity
IMMUNOMETHODS 1, 177-183 (1992)
Cytokine MAPPing in Autoirnrnunity David B. Wilde,* Dennis M. Klinman,* and Steven R. Bauer ~ *Laboratory of Retroviral Immunology and tLaboratory of Molecular Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892
The polymerase chain reaction is a powerful tool for deriving semi-quantitative information about the relative abundance of cytokine RNA species that are present in pathologic states, such as autoimmune disease. We describe here the MAPPing technique, by which multiple cytokine RNA species can be analyzed from small tissue samples. A revealing array of information concerning the transcriptional activity of cells can be obtained with this technique. We discuss the qualitative and quantitative aspects of MAPPing that should be considered for optimal performance of the assay and for a constructive interpretation of the results. © 1992AcademicPress,Inc.
Autoimmunity is recognized as the cause of an increasing number of pathologic states. It can be induced by a cross-reactive response to foreign/xenoantigens, by the recognition of self-antigens at previously privileged anatomical sites, or by a breakdown in self-tolerance. Over the past three decades, the characteristic cellular abnormalities associated with the pathogenesis of autoimmune disease have been studied extensively. With the advent of increasingly sophisticated techniques in molecular biology, a more comprehensive assessment of the mechanisms and etiology of autoimmunity can now be achieved. For example, a number of investigators have shown that locally acting cytokines play a major role in regulating the complex processes in the generation and perpetuation of an autoimmune response. Many cytokines capable of modulating the proliferation and/ or differentiation of potentially autoreactive cells have been identified by in vitro studies of cell populations loss-66sT/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in a n y form reserved.
(1, 2). For example, IFN-~ was found to induce a striking increase in IgG2a secretion by murine B cells (35), while IL-4 up-regulated their production of IgG1 (6-8). The cytokine IL-6 has been detected in the serum of normal individuals, and abnormalities in IL-6 levels are associated with diseases of the immune system such as SLE, psoriasis, and rheumatoid arthritis (9-11). The influence of such cytokines was initially assessed by adding them to cell cultures, with or without anticytokine antibodies (12, 13). By this method, a selective increase in the production of IgG anti-DNA autoantibodies was induced by treating autoimmune NZB/W B cells with IL-5 and MRL/Ipr B cells with IFN-~ (1416). Yet this approach did not establish whether the production of such cytokines was abnormal in vivo. To that end, molecular techniques have been used to examine the levels of cytokine messenger RNA (mRNA) in normal and autoimmune animals. One of the more widely used tools for exploring cytokine gene expression is the polymerase chain reaction (PCR) (17-22). Briefly, PCR is a technique for amplifying a target DNA sequence (or the complementary cDNA for RNA species) in vitro by repeated rounds of thermal denaturation of double-stranded DNA, annealing short oligonucleotide primers that are complementary and specific to the DNA region to be amplified, and then enzymatically extending the primer sequence to generate from 105 to 109 copies of the targeted sequence. A thermostable DNA polymerase, Taq, is used to maintain enzymatic activity during the many cycles of PCR amplification. Many reviews and protocols that give the exact laboratory methodology of PCR have been written, and the reader who is interested in utilizing PCR for the first time is encouraged to use these sources before attempting more complex PCR appli177
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cations (18, 23). By using PCR as the basis for the development of specialized techniques, cytokine MAPPing (Message Amplification Phenotyping) has evolved (24). The latter approach involves the simultaneous amplification of cDNAs derived from mRNAs encoding a variety of cytokines from a small number of cells by using multiple primer pairs. This technique allows semi-quantitative measurement of transcriptional activity among a large array of gene transcripts (Fig. 1). In the context of the present work, MAPPing is used to measure the relative amount of cytokine message produced in animals with autoimmune disease. MAPPing utilizes the same materials and laboratory equipment as those used for PCR. To analyze a tissue sample for the activity of multiple cytokines, one must have primer pairs for each cytokine to be studied. In studies in which the cytokine of interest is not known a priori, it is not uncommon to test 20 or more primer pairs by MAPPing to assess rapidly the cytokine mRNAs that are present. First, RNA is isolated from tissue, usually by extraction of cytoplasmic RNA (25) or by acid phenol/chloroform extraction of total RNA (26). Second, RNA is converted to cDNA to prepare it for PCR amplification; the choice of technique for this
oe
• • • • •
o •
• • •
SINGLE PRIMER PCR
MAPPing PCR
EXTRACTDNA/RNA FROM CELLS
MULTIPLEX PCR
FIG. 1. Schematic representation of different techniques of PCR analysis. Following extraction of RNA from cells, cDNA template is amplified by PCR using (from left to right): a single cytokine primer pair to generate one band corresponding to the amplified segment for that cytokine; one cytokine primer pair in each PCR tube, but with simultaneous amplification of multiple tubes to yield a multilane gel with each amplified segment representing the presence of a different cytokine (MAPPing); or multiple cytokine pairs in a single PCR tube to simultaneously amplify cDNA species (multiplex PCR), as depicted by multiple amplified bands present within a single gel lane.
step is discussed later. Finally, cDNA is amplified by multiple primer pairs, with each pair specific for a particular cytokine, and the amplified products are then visualized as multiple bands on a gel, probed, or eluted for further analysis. To clarify a point of nomenclature, MAPPing originally referred to amplification of each primer pair within a separate PCR reaction tube, with the use of a single, uniform set of temperatures for denaturation, annealing, and extension (24). When multiple primers are added into a single PCR tube, this technique is referred to as multiplex PCR (27), but primer pairs are amplified with a single uniform temperature profile, as is done in MAPPing. The essential difference between MAPPing and multiplex PCR is the number of primers that are added per individual PCR tube, but both techniques are used for the simultaneous analysis of a template by multiple primer pairs. For simplicity, we include together technical comments on both MAPPing and multiplex PCR. The usefulness of cytokine MAPPing as a strategy for measuring the relative production of various mRNA species is constrained by the needs of the investigator. Considerations include (i) the sensitivity of detection required, (ii) the amount of tissue available for analysis, (iii) the number of samples to be processed, (iv) the number of cytokines to be analyzed, (v) the need to correlate mRNA concentration with concentration of secreted protein, and (vi) the need to assess cytokine mRNA levels in small numbers of cells. Technical aspects of MAPPing can lead to systematic errors of quantitation, even when internal standards from control specimens are included. Therefore, as with data from other specialized molecular biological techniques, those obtained from cytokine MAPPing must be interpreted with care. Historically, bioassays were used to distinguish alterations in cytokine production among autoreactive and normal cell populations. However, such assays are subject to a number of pitfalls. First, the detection of cytokines was limited to those proteins for which a responsive cell line was available. Short-lived cytokines were subject to degradation prior to assay. Since many cell lines proliferate to several different cytokines, the specificity of such bioassays is reduced. Moreover, sensitivity varied from one line to the next, introducing problems in the comparison of cytokine titers obtained using different cell lines. Some cytokines, such as IL2, can be consumed by the cell population being tested, so bioassays for IL-2 are only able to measure differences between cytokine production and consumption.
CYTOKINE MAPPing IN AUTOIMMUNITY For these reasons, a more quantitative approach to measurement of cytokine production was needed. Analysis of cytokine production using techniques from molecular biology has provided insights that would have been difficult to obtain at the cellular level. For instance, cytokines such as I L - l a and IL-I~ were found to differ greatly in amino acid composition, but exerted similar effects on cells through their common use of the IL-1 receptor (28). Likewise, even cytokines with a high degree of homology, such as those of the colony-stimulating family, are readily distinguished by PCR but distinct target cell populations are required to quantitate their biological activity in vitro (29). Sequence analysis of PCR-amplified cytokine mRNA also can reveal evolutionary homology and yield insights about the potential function of cytokines that show close relationship. Indeed, the grouping of cytokines into family trees has been made possible primarily by analysis of their molecular characteristics rather than by observation of their behavior in cell culture systems (30). Nevertheless, data generated using PCR must be interpreted with caution. Any method that does not directly measure a protein's biological activity can erroneously detect message or product that is nonfunctional in uivo. This concern is equally pertinent to antibody-based detection systems (ELISA) and to assays designed to quantify mRNA. In either case, the protein of interest may not be produced in an active form, secondary to degradation, lack of translation, defective post-translational modification, or faulty transport to the cell surface. Limited data suggest that IL-2 mRNA levels correspond roughly to the concentration of secreted IL-2, such that differences in IL-2 titers between cell populations reflect variability in IL2 mRNA production by those cells (31). However, the concentration of other cytokines, such as IL-1/3, do not appear to reflect message levels. It is believed that endotoxin present in fetal calf serum may be responsible for both inducing and altering the half-lives of IL-1/3 and other cytokines (32). Complex interactions can regulate the level of cytokine mRNA within a cell. It is known, for instance, that a conserved AU consensus sequence downstream of the 3' end of certain cytokine mRNAs acts as a binding site for elements that contribute to the degradation of message and that this sequence is stabilized following treatment of cells with phorbol esters or antibodies to the cell surface determinant CD28 (33). As noted above, the presence of endotoxin can artificially induce cytokine message following brief exposure of cells to serum, and in addition, both production and stabili-
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zation of certain cytokine mRNAs are influenced by the concentration of other mRNAs within the cytoplasm (34). Consequently, there is ample opportunity to detect artifacts of cytokine mRNA production, and therefore we conclude that quantitation of cytokine message, although precise, is subject to many of the errors of interpretation that plague its counterpart, the bioassay. Perhaps the best solution to this dilemma is to combine assay techniques, with special attention to experimental designs that minimize false-positive production of cytokines through cell manipulation. For this reason, speedy processing of cells e x viuo is essential (35). Given the caveats mentioned above, the analysis of cytokine mRNA using PCR and MAPPing technology can be very revealing. When RNA from autoreactive cells is abundant, sensitivity sufficient for detection of message can be achieved by Northern gel or dot blot analysis (36-41). Rapid processing of multiple samples can be accomplished by cell lysis with a nonionic detergent, such as NP-40, and total cytoplasmic RNA can be blotted and probed in short order. Detection of rare message is limited primarily by cell number, efficiency of blotting (noncharged nylon membranes are generally believed to give a more consistent and higher quality signal than nitrocellulose filters) (42), and probe length. A drawback to Northern analysis is a relative lack of sensitivity compared to methods that use amplification by PCR, such as cytokine MAPPing. This can be overcome by amplifying the mRNA by PCR after cDNA production. If this route is chosen, though, the investigator must be aware that measurements of cytokine message may be less accurate, as is discussed below, because experimental errors introduced by differences in the efficiency of blotting and probing occur only once at each step of Northern analysis, whereas efficiency differences in PCR amplification occur with a compounding effect during each cycle. Cytokine MAPPing utilizes the same experimental principles as Northern analysis, but has been developed to accommodate special experimental conditions, namely, small sample size (a typical analysis uses one million cells or less), compact design which allows multiple PCR amplifications to occur simultaneously on a single tissue sample, and rapid processing of tissue so that kinetic studies generally can be accomplished within the same day. The procedure requires isolation of RNA, followed by reverse transcription to cDNA and amplification by PCR. Amplified products can be visualized directly on a gel or probed by standard techniques. The following technical considerations should be considered to optimize MAPPing strategy.
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TECHNICAL CONSIDERATIONS Tissue Tissue may be obtained from virtually any source, but care should be taken to process it rapidly. In particular, samples must be preserved in a detergent-containing solution that inhibits the degradation of RNA. Cytokine mRNAs (unlike cell-associated proteins) can degrade rapidly in nonphysiologic environments. We recommend lysis in NP-40 solution for extraction of cytoplasmic RNA or extraction of total RNA by dissolution of cells in Solution D, a formulation that contains guanidinium thiocyanate, a potent RNase inhibitor, and n-lauroylsarcosine, an ionic detergent (26). Another possibility is to snap-freeze cells in liquid nitrogen. It is also generally desirable to remove erythrocytes from tissue preparations, but gradient separation can be time-consuming and cumbersome and can permit the degradation of mRNA. Hypotonic lysis followed by cell pelleting will suffice to decrease unwanted protein contamination (43). RNA preparation by cesium chloride gradient centrifugation has been replaced largely by more rapid techniques using acid phenol/chloroform extraction mixtures, but is still a reliable method of obtaining RNA of high purity. Passage over an oligo(dT) column purifies mRNA selectively, but for most applications, it is adequate to use total RNA in PCR amplification. Interestingly, it has been reported that proteinase K digestion can proceed in the presence of 4 M gnanidinium thiocyanate (Solution D), thereby allowing the investigator to split the sample and purify DNA directly or continue with phenol/chloroform extraction to isolate total RNA. The use of ionic detergents in the preparation of cDNA/DNA for PCR can create problems because these detergents can also solubilize Taq polymerase and thereby decrease its enzymatic efficiency. Taq is quite sensitive to the effects of ionic detergents such as SDS or Sarcosyl, and standard buffer preparations containing these detergents must be diluted up to 100-fold to eliminate inhibitory effects (44). Nonionic detergents, such as NP-40 and Tween 20, do not denature Taq, and thus microscale preparations with nonionic detergents are less likely to interfere with efficient amplification during cytokine MAPPing.
Primer Selection Customized or commercial primer pairs are suitable for cytokine MAPPing. Conventional rules for selecting primers apply to cytokine MAPPing. One should (i)
avoid complementarity at the 3' ends to prevent primerdimer artifacts; (ii) avoid sequences with internal complementarity, secondary structure, or repetitive stretches of one nucleotide; (iii) use primers with G or C terminal nucleotides, if possible, to increase the probability of firm 3' annealing; (iv) select exon-spanning sequences to distinguish amplified RNA from genomic DNA; and (v) select 5' and 3' primer sequences with similar G-FC content so that annealing conditions will be optimal for both primers. Because first-strand cDNA is the antisense strand, the 3' PCR primer sequence must correspond to the sense-strand sequence. Special considerations for selecting primers useful for MAPPing include the use of primers with similar G+C content for all the genes to be measured simultaneously and the use of primers that amplify PCR products that are close in size, yet resolvable by the gel separation system, in order to generate easily distinguishable products while avoiding length-based efficiency differences of PCR amplification. Several considerations in multiplex PCR are unique to the simultaneous use of multiple primers in a single PCR tube. Critical parameters of melting point, optimal magnesium concentration, and 3' complementarity in MAPPing can differ from those in conventional PCR. Multiple primers can compete for template, magnesium ion, nucleotides, and Taq polymerase in later cycles of amplification. Before use in MAPPing, each primer set should be tested independently in the absence of other primers to determine optimal conditions for amplification with a high degree of specificity. Differences in amplification efficiency during MAPPing can be minimized by attention to certain details: (i) use of primers with a G+C content that does not exceed 60%, since the kinetics of melting and annealing may not be optimal for such primers at the single temperature point that must be used for all primers present in the MAPPing experiment; (ii) use of primers that amplify gene sequences of approximately the same length (within several hundred nucleotides), especially if two-temperature PCR is used and ramp time may become a rate-limiting factor for extension of product as nucleotides become scarce in later cycles of amplification; (iii) use of random hexamer primers during first-strand cDNA synthesis to avoid bias toward the 3' end of mRNAs (if oligo(dT) primers are used, PCR primers should be at approximately equal distance from the oligo(dT) priming site); and (iv) use of a magnesium ion concentration that is slightly in excess of that used for optimal amplification of single primer set PCR. Recombinant Taq polymerase that has stable high activity over magnesium concentrations between 2 and
CYTOKINE MAPPing IN AUTOIMMUNITY 10 mM is commercially available and is recommended for MAPPing, where magnesium concentrations as high as 5 mM may be required to generate the greatest quantity of PCR product in the presence of multiple primers (27). Again, optimal magnesium concentration is derived empirically, as too much magnesium tends to generate shortened PCR products and an excessive number of bands at low molecular weights (45). If a PCR band is absent during MAPPing but present when cDNA is amplified by the same primer pair used in single primer PCR, it is likely that competition among cDNA species is preventing efficient amplification. Some primer pairs are "selfish," in that they generate PCR product much more efficiently than other cDNA species. To resolve this problem, one can either use a different primer set for the dominant species or perform MAPPing selectively by deleting this primer pair from the reaction. Absent bands during MAPPing also may be due to low magnesium concentration or limiting quantities of nucleotides or Taq. We recommend a nucleotide concentration in the range 100-200 #M and 2 or 2.5 units of Taq per 100-t~l PCR reaction volume. The multiplicity of bands generated by MAPPing necessitates the removal of spurious bands from the final product. Removal of genomic DNA is a first step toward the amplification of a pure RNA preparation. Optimal magnesium concentration can prevent the accumulation of truncated PCR products, and selection of higher annealing temperature generally diminishes nonspecific template priming. Smearing of bands can occur from excessive cycle number, resulting in amplification of background "noise," from prolonged annealing and extension times, from excessive Taq or magnesium, or simply from poor gel resolution (45). Some primer pairs have persistent companion bands despite all precaution. In this case, it is better to use an alternative primer pair or nested pairs of primers to eliminate the unwanted species. Several innovative techniques for improving specificity of RNA amplification have been described, using variations in primer selection or cDNA production (46-49). In general, however, these techniques have been developed for amplification of a single RNA species and may not be practical for MAPPing.
Efficiency An extremely important issue in judging relative RNA expression using PCR is the efficiency of PCR amplification for the various templates. Lack of consideration of this issue can lead to misinterpretation of PCR data. The crux of this issue is whether or not
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PCR efficiencies for a given template are equal for PCR reactions performed on different samples, in different tubes containing the same samples, and at different times. For the MAPPing procedure to yield accurate data, the amplification efficiencies for a given template should be equal in the different samples that are being compared. If the reaction efficiencies are the same, the amount of PCR product generated from each sample is proportional to the original amount of the particular cDNA. In Northern blotting analysis of relative gene expression, it is often assumed that a reference gene, such as ~-actin or GAPDH, is expressed at equal levels in RNA from a variety of sources. Demonstration of equal hybridization signals to actin or GAPDH RNA from different samples is thus used as a reference for comparing levels of hybridization for other genes such as cytokines. Semi-quantitative PCR uses the same assumption of equal expression of a reference gene among samples from a variety of sources. In both cases, inaccurate data can arise from problems inherent in the technique. In Northern blotting, the specific activity and hybridization kinetics of the different probes are the primary sources of experimental error. In PCR, the primary inherent source of experimental error is the efficiency of amplification. Since these errors are compounded in each PCR amplification cycle, they can be sources of significant error. The same primer pair can have different PCR efficiencies when cDNAs derived from different sources are being used, although the same sequence is being amplified. This probably results from organic or metal ion contaminants present in the RNA preparation used to generate cDNA for PCR amplification. By monitoring PCR product formation through multiple rounds of PCR cycles, termed PCR cycle titration, one can assess the efficiency of amplification for a particular template. Using these cycle titration measurements, one can establish whether a particular RNA such as ~-actin is actually expressed at similar levels in two different RNA sources. If so, one could then compare the abundance of a second PCR template in units relative to ~-actin, provided t hat the efficiency of the second template was also equal in the two samples (50). T he efficiency of PCR can be determined by using radioactive PCR primers and quantifying the PCR product generated after differing cycle numbers. Plotting the log of the PCR product versus the cycle number will generate a straight line if measurements are taken during the exponential phase of the PCR reaction (Fig. 2). The slope of the line will be determined by the PC R efficiency. If the slopes are the same in a comparison of amplification of the same template in two different
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WILDE, KLINMAN,
samples, the efficiencies of the PC R reactions are the same. One final caveat to this technique is the underlying assumption that the reverse transcription reaction efficiency is the same for the RNA samples being compared. The use of internal RNA standards present in the cDNA synthesis and subsequent PCR reaction can overcome this problem, as discussed elsewhere (50). Because the parameters for PCR amplification t hat are chosen for any particular MAPPing experiment are unlikely to be optimal for all primer pairs t hat are present, it is especially important to remember t hat the intensity of bands for different cytokines does not directly correlate with absolute mRNA concentration, because the efficiency of amplification for each cytokine mRNA is different and is compounded with each cycle of amplification. Our recommendation for garnering useful and accurate information about cytokine expression by PCR would be to screen samples of interest by MAPPing. If intriguing differences are found in expression of a few genes, these differences should be investigated carefully by PCR cycle titration techniques as outlined above. T he possibility t hat interesting differences would be missed or subject to misinterpretation due to artifact would thus be minimized.
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_ IG. 2. Results of three experiments, A, B, and C, using the same set of primers. Experiments A and B resulted in cycle titrations with equal slopes, but different y intercepts. Therefore, these two experiments can be compared and show t h a t there is approximately a fivefold difference in the abundance of the cDNA being measured. Experiment C has a slope different from that of either of the other two experiments, and thus data from this cannot be used to estimate relative amounts of this cDNA. Experiments A and B can be compared quantitatively only if these two samples have superimposed lines from a reference such as fi-actin which resulted from the same initial input amount of RNA.
AND BAUER
CONCLUSION In conclusion, cytokine MAPPing (or MAPPing of any family of related gene products) can yield semiquantitative data concerning relative proportions of RNA species that are present in activated cells. Its principal asset is the ability to extract rapidly a large amount of information about simultaneous transcriptional events that are so numerous or that occur in such a rare population of cells that repetitive applications of single primer PCR would be prohibitive. The interpretation of MAPPing results is subject to the same types of precautions that apply to single primer PCR, and it is always advisable to seek alternative methods to verify MAPPing results. Nevertheless, MAPPing is a versatile addition to a growing number of PCR applications that can be used routinely to explore complex physiology in pathologic states.
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CYTOKINE MAPPing IN AUTOIMMUNITY
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Cytokine MAPPing in Autoirnrnunity David B. Wilde,* Dennis M. Klinman,* and Steven R. Bauer ~ *Laboratory of Retroviral Immunology and tLaboratory of Molecular Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892
The polymerase chain reaction is a powerful tool for deriving semi-quantitative information about the relative abundance of cytokine RNA species that are present in pathologic states, such as autoimmune disease. We describe here the MAPPing technique, by which multiple cytokine RNA species can be analyzed from small tissue samples. A revealing array of information concerning the transcriptional activity of cells can be obtained with this technique. We discuss the qualitative and quantitative aspects of MAPPing that should be considered for optimal performance of the assay and for a constructive interpretation of the results. © 1992AcademicPress,Inc.
Autoimmunity is recognized as the cause of an increasing number of pathologic states. It can be induced by a cross-reactive response to foreign/xenoantigens, by the recognition of self-antigens at previously privileged anatomical sites, or by a breakdown in self-tolerance. Over the past three decades, the characteristic cellular abnormalities associated with the pathogenesis of autoimmune disease have been studied extensively. With the advent of increasingly sophisticated techniques in molecular biology, a more comprehensive assessment of the mechanisms and etiology of autoimmunity can now be achieved. For example, a number of investigators have shown that locally acting cytokines play a major role in regulating the complex processes in the generation and perpetuation of an autoimmune response. Many cytokines capable of modulating the proliferation and/ or differentiation of potentially autoreactive cells have been identified by in vitro studies of cell populations loss-66sT/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in a n y form reserved.
(1, 2). For example, IFN-~ was found to induce a striking increase in IgG2a secretion by murine B cells (35), while IL-4 up-regulated their production of IgG1 (6-8). The cytokine IL-6 has been detected in the serum of normal individuals, and abnormalities in IL-6 levels are associated with diseases of the immune system such as SLE, psoriasis, and rheumatoid arthritis (9-11). The influence of such cytokines was initially assessed by adding them to cell cultures, with or without anticytokine antibodies (12, 13). By this method, a selective increase in the production of IgG anti-DNA autoantibodies was induced by treating autoimmune NZB/W B cells with IL-5 and MRL/Ipr B cells with IFN-~ (1416). Yet this approach did not establish whether the production of such cytokines was abnormal in vivo. To that end, molecular techniques have been used to examine the levels of cytokine messenger RNA (mRNA) in normal and autoimmune animals. One of the more widely used tools for exploring cytokine gene expression is the polymerase chain reaction (PCR) (17-22). Briefly, PCR is a technique for amplifying a target DNA sequence (or the complementary cDNA for RNA species) in vitro by repeated rounds of thermal denaturation of double-stranded DNA, annealing short oligonucleotide primers that are complementary and specific to the DNA region to be amplified, and then enzymatically extending the primer sequence to generate from 105 to 109 copies of the targeted sequence. A thermostable DNA polymerase, Taq, is used to maintain enzymatic activity during the many cycles of PCR amplification. Many reviews and protocols that give the exact laboratory methodology of PCR have been written, and the reader who is interested in utilizing PCR for the first time is encouraged to use these sources before attempting more complex PCR appli177
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WILDE, KLINMAN, AND BAUER
cations (18, 23). By using PCR as the basis for the development of specialized techniques, cytokine MAPPing (Message Amplification Phenotyping) has evolved (24). The latter approach involves the simultaneous amplification of cDNAs derived from mRNAs encoding a variety of cytokines from a small number of cells by using multiple primer pairs. This technique allows semi-quantitative measurement of transcriptional activity among a large array of gene transcripts (Fig. 1). In the context of the present work, MAPPing is used to measure the relative amount of cytokine message produced in animals with autoimmune disease. MAPPing utilizes the same materials and laboratory equipment as those used for PCR. To analyze a tissue sample for the activity of multiple cytokines, one must have primer pairs for each cytokine to be studied. In studies in which the cytokine of interest is not known a priori, it is not uncommon to test 20 or more primer pairs by MAPPing to assess rapidly the cytokine mRNAs that are present. First, RNA is isolated from tissue, usually by extraction of cytoplasmic RNA (25) or by acid phenol/chloroform extraction of total RNA (26). Second, RNA is converted to cDNA to prepare it for PCR amplification; the choice of technique for this
oe
• • • • •
o •
• • •
SINGLE PRIMER PCR
MAPPing PCR
EXTRACTDNA/RNA FROM CELLS
MULTIPLEX PCR
FIG. 1. Schematic representation of different techniques of PCR analysis. Following extraction of RNA from cells, cDNA template is amplified by PCR using (from left to right): a single cytokine primer pair to generate one band corresponding to the amplified segment for that cytokine; one cytokine primer pair in each PCR tube, but with simultaneous amplification of multiple tubes to yield a multilane gel with each amplified segment representing the presence of a different cytokine (MAPPing); or multiple cytokine pairs in a single PCR tube to simultaneously amplify cDNA species (multiplex PCR), as depicted by multiple amplified bands present within a single gel lane.
step is discussed later. Finally, cDNA is amplified by multiple primer pairs, with each pair specific for a particular cytokine, and the amplified products are then visualized as multiple bands on a gel, probed, or eluted for further analysis. To clarify a point of nomenclature, MAPPing originally referred to amplification of each primer pair within a separate PCR reaction tube, with the use of a single, uniform set of temperatures for denaturation, annealing, and extension (24). When multiple primers are added into a single PCR tube, this technique is referred to as multiplex PCR (27), but primer pairs are amplified with a single uniform temperature profile, as is done in MAPPing. The essential difference between MAPPing and multiplex PCR is the number of primers that are added per individual PCR tube, but both techniques are used for the simultaneous analysis of a template by multiple primer pairs. For simplicity, we include together technical comments on both MAPPing and multiplex PCR. The usefulness of cytokine MAPPing as a strategy for measuring the relative production of various mRNA species is constrained by the needs of the investigator. Considerations include (i) the sensitivity of detection required, (ii) the amount of tissue available for analysis, (iii) the number of samples to be processed, (iv) the number of cytokines to be analyzed, (v) the need to correlate mRNA concentration with concentration of secreted protein, and (vi) the need to assess cytokine mRNA levels in small numbers of cells. Technical aspects of MAPPing can lead to systematic errors of quantitation, even when internal standards from control specimens are included. Therefore, as with data from other specialized molecular biological techniques, those obtained from cytokine MAPPing must be interpreted with care. Historically, bioassays were used to distinguish alterations in cytokine production among autoreactive and normal cell populations. However, such assays are subject to a number of pitfalls. First, the detection of cytokines was limited to those proteins for which a responsive cell line was available. Short-lived cytokines were subject to degradation prior to assay. Since many cell lines proliferate to several different cytokines, the specificity of such bioassays is reduced. Moreover, sensitivity varied from one line to the next, introducing problems in the comparison of cytokine titers obtained using different cell lines. Some cytokines, such as IL2, can be consumed by the cell population being tested, so bioassays for IL-2 are only able to measure differences between cytokine production and consumption.
CYTOKINE MAPPing IN AUTOIMMUNITY For these reasons, a more quantitative approach to measurement of cytokine production was needed. Analysis of cytokine production using techniques from molecular biology has provided insights that would have been difficult to obtain at the cellular level. For instance, cytokines such as I L - l a and IL-I~ were found to differ greatly in amino acid composition, but exerted similar effects on cells through their common use of the IL-1 receptor (28). Likewise, even cytokines with a high degree of homology, such as those of the colony-stimulating family, are readily distinguished by PCR but distinct target cell populations are required to quantitate their biological activity in vitro (29). Sequence analysis of PCR-amplified cytokine mRNA also can reveal evolutionary homology and yield insights about the potential function of cytokines that show close relationship. Indeed, the grouping of cytokines into family trees has been made possible primarily by analysis of their molecular characteristics rather than by observation of their behavior in cell culture systems (30). Nevertheless, data generated using PCR must be interpreted with caution. Any method that does not directly measure a protein's biological activity can erroneously detect message or product that is nonfunctional in uivo. This concern is equally pertinent to antibody-based detection systems (ELISA) and to assays designed to quantify mRNA. In either case, the protein of interest may not be produced in an active form, secondary to degradation, lack of translation, defective post-translational modification, or faulty transport to the cell surface. Limited data suggest that IL-2 mRNA levels correspond roughly to the concentration of secreted IL-2, such that differences in IL-2 titers between cell populations reflect variability in IL2 mRNA production by those cells (31). However, the concentration of other cytokines, such as IL-1/3, do not appear to reflect message levels. It is believed that endotoxin present in fetal calf serum may be responsible for both inducing and altering the half-lives of IL-1/3 and other cytokines (32). Complex interactions can regulate the level of cytokine mRNA within a cell. It is known, for instance, that a conserved AU consensus sequence downstream of the 3' end of certain cytokine mRNAs acts as a binding site for elements that contribute to the degradation of message and that this sequence is stabilized following treatment of cells with phorbol esters or antibodies to the cell surface determinant CD28 (33). As noted above, the presence of endotoxin can artificially induce cytokine message following brief exposure of cells to serum, and in addition, both production and stabili-
1"79
zation of certain cytokine mRNAs are influenced by the concentration of other mRNAs within the cytoplasm (34). Consequently, there is ample opportunity to detect artifacts of cytokine mRNA production, and therefore we conclude that quantitation of cytokine message, although precise, is subject to many of the errors of interpretation that plague its counterpart, the bioassay. Perhaps the best solution to this dilemma is to combine assay techniques, with special attention to experimental designs that minimize false-positive production of cytokines through cell manipulation. For this reason, speedy processing of cells e x viuo is essential (35). Given the caveats mentioned above, the analysis of cytokine mRNA using PCR and MAPPing technology can be very revealing. When RNA from autoreactive cells is abundant, sensitivity sufficient for detection of message can be achieved by Northern gel or dot blot analysis (36-41). Rapid processing of multiple samples can be accomplished by cell lysis with a nonionic detergent, such as NP-40, and total cytoplasmic RNA can be blotted and probed in short order. Detection of rare message is limited primarily by cell number, efficiency of blotting (noncharged nylon membranes are generally believed to give a more consistent and higher quality signal than nitrocellulose filters) (42), and probe length. A drawback to Northern analysis is a relative lack of sensitivity compared to methods that use amplification by PCR, such as cytokine MAPPing. This can be overcome by amplifying the mRNA by PCR after cDNA production. If this route is chosen, though, the investigator must be aware that measurements of cytokine message may be less accurate, as is discussed below, because experimental errors introduced by differences in the efficiency of blotting and probing occur only once at each step of Northern analysis, whereas efficiency differences in PCR amplification occur with a compounding effect during each cycle. Cytokine MAPPing utilizes the same experimental principles as Northern analysis, but has been developed to accommodate special experimental conditions, namely, small sample size (a typical analysis uses one million cells or less), compact design which allows multiple PCR amplifications to occur simultaneously on a single tissue sample, and rapid processing of tissue so that kinetic studies generally can be accomplished within the same day. The procedure requires isolation of RNA, followed by reverse transcription to cDNA and amplification by PCR. Amplified products can be visualized directly on a gel or probed by standard techniques. The following technical considerations should be considered to optimize MAPPing strategy.
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TECHNICAL CONSIDERATIONS Tissue Tissue may be obtained from virtually any source, but care should be taken to process it rapidly. In particular, samples must be preserved in a detergent-containing solution that inhibits the degradation of RNA. Cytokine mRNAs (unlike cell-associated proteins) can degrade rapidly in nonphysiologic environments. We recommend lysis in NP-40 solution for extraction of cytoplasmic RNA or extraction of total RNA by dissolution of cells in Solution D, a formulation that contains guanidinium thiocyanate, a potent RNase inhibitor, and n-lauroylsarcosine, an ionic detergent (26). Another possibility is to snap-freeze cells in liquid nitrogen. It is also generally desirable to remove erythrocytes from tissue preparations, but gradient separation can be time-consuming and cumbersome and can permit the degradation of mRNA. Hypotonic lysis followed by cell pelleting will suffice to decrease unwanted protein contamination (43). RNA preparation by cesium chloride gradient centrifugation has been replaced largely by more rapid techniques using acid phenol/chloroform extraction mixtures, but is still a reliable method of obtaining RNA of high purity. Passage over an oligo(dT) column purifies mRNA selectively, but for most applications, it is adequate to use total RNA in PCR amplification. Interestingly, it has been reported that proteinase K digestion can proceed in the presence of 4 M gnanidinium thiocyanate (Solution D), thereby allowing the investigator to split the sample and purify DNA directly or continue with phenol/chloroform extraction to isolate total RNA. The use of ionic detergents in the preparation of cDNA/DNA for PCR can create problems because these detergents can also solubilize Taq polymerase and thereby decrease its enzymatic efficiency. Taq is quite sensitive to the effects of ionic detergents such as SDS or Sarcosyl, and standard buffer preparations containing these detergents must be diluted up to 100-fold to eliminate inhibitory effects (44). Nonionic detergents, such as NP-40 and Tween 20, do not denature Taq, and thus microscale preparations with nonionic detergents are less likely to interfere with efficient amplification during cytokine MAPPing.
Primer Selection Customized or commercial primer pairs are suitable for cytokine MAPPing. Conventional rules for selecting primers apply to cytokine MAPPing. One should (i)
avoid complementarity at the 3' ends to prevent primerdimer artifacts; (ii) avoid sequences with internal complementarity, secondary structure, or repetitive stretches of one nucleotide; (iii) use primers with G or C terminal nucleotides, if possible, to increase the probability of firm 3' annealing; (iv) select exon-spanning sequences to distinguish amplified RNA from genomic DNA; and (v) select 5' and 3' primer sequences with similar G-FC content so that annealing conditions will be optimal for both primers. Because first-strand cDNA is the antisense strand, the 3' PCR primer sequence must correspond to the sense-strand sequence. Special considerations for selecting primers useful for MAPPing include the use of primers with similar G+C content for all the genes to be measured simultaneously and the use of primers that amplify PCR products that are close in size, yet resolvable by the gel separation system, in order to generate easily distinguishable products while avoiding length-based efficiency differences of PCR amplification. Several considerations in multiplex PCR are unique to the simultaneous use of multiple primers in a single PCR tube. Critical parameters of melting point, optimal magnesium concentration, and 3' complementarity in MAPPing can differ from those in conventional PCR. Multiple primers can compete for template, magnesium ion, nucleotides, and Taq polymerase in later cycles of amplification. Before use in MAPPing, each primer set should be tested independently in the absence of other primers to determine optimal conditions for amplification with a high degree of specificity. Differences in amplification efficiency during MAPPing can be minimized by attention to certain details: (i) use of primers with a G+C content that does not exceed 60%, since the kinetics of melting and annealing may not be optimal for such primers at the single temperature point that must be used for all primers present in the MAPPing experiment; (ii) use of primers that amplify gene sequences of approximately the same length (within several hundred nucleotides), especially if two-temperature PCR is used and ramp time may become a rate-limiting factor for extension of product as nucleotides become scarce in later cycles of amplification; (iii) use of random hexamer primers during first-strand cDNA synthesis to avoid bias toward the 3' end of mRNAs (if oligo(dT) primers are used, PCR primers should be at approximately equal distance from the oligo(dT) priming site); and (iv) use of a magnesium ion concentration that is slightly in excess of that used for optimal amplification of single primer set PCR. Recombinant Taq polymerase that has stable high activity over magnesium concentrations between 2 and
CYTOKINE MAPPing IN AUTOIMMUNITY 10 mM is commercially available and is recommended for MAPPing, where magnesium concentrations as high as 5 mM may be required to generate the greatest quantity of PCR product in the presence of multiple primers (27). Again, optimal magnesium concentration is derived empirically, as too much magnesium tends to generate shortened PCR products and an excessive number of bands at low molecular weights (45). If a PCR band is absent during MAPPing but present when cDNA is amplified by the same primer pair used in single primer PCR, it is likely that competition among cDNA species is preventing efficient amplification. Some primer pairs are "selfish," in that they generate PCR product much more efficiently than other cDNA species. To resolve this problem, one can either use a different primer set for the dominant species or perform MAPPing selectively by deleting this primer pair from the reaction. Absent bands during MAPPing also may be due to low magnesium concentration or limiting quantities of nucleotides or Taq. We recommend a nucleotide concentration in the range 100-200 #M and 2 or 2.5 units of Taq per 100-t~l PCR reaction volume. The multiplicity of bands generated by MAPPing necessitates the removal of spurious bands from the final product. Removal of genomic DNA is a first step toward the amplification of a pure RNA preparation. Optimal magnesium concentration can prevent the accumulation of truncated PCR products, and selection of higher annealing temperature generally diminishes nonspecific template priming. Smearing of bands can occur from excessive cycle number, resulting in amplification of background "noise," from prolonged annealing and extension times, from excessive Taq or magnesium, or simply from poor gel resolution (45). Some primer pairs have persistent companion bands despite all precaution. In this case, it is better to use an alternative primer pair or nested pairs of primers to eliminate the unwanted species. Several innovative techniques for improving specificity of RNA amplification have been described, using variations in primer selection or cDNA production (46-49). In general, however, these techniques have been developed for amplification of a single RNA species and may not be practical for MAPPing.
Efficiency An extremely important issue in judging relative RNA expression using PCR is the efficiency of PCR amplification for the various templates. Lack of consideration of this issue can lead to misinterpretation of PCR data. The crux of this issue is whether or not
181
PCR efficiencies for a given template are equal for PCR reactions performed on different samples, in different tubes containing the same samples, and at different times. For the MAPPing procedure to yield accurate data, the amplification efficiencies for a given template should be equal in the different samples that are being compared. If the reaction efficiencies are the same, the amount of PCR product generated from each sample is proportional to the original amount of the particular cDNA. In Northern blotting analysis of relative gene expression, it is often assumed that a reference gene, such as ~-actin or GAPDH, is expressed at equal levels in RNA from a variety of sources. Demonstration of equal hybridization signals to actin or GAPDH RNA from different samples is thus used as a reference for comparing levels of hybridization for other genes such as cytokines. Semi-quantitative PCR uses the same assumption of equal expression of a reference gene among samples from a variety of sources. In both cases, inaccurate data can arise from problems inherent in the technique. In Northern blotting, the specific activity and hybridization kinetics of the different probes are the primary sources of experimental error. In PCR, the primary inherent source of experimental error is the efficiency of amplification. Since these errors are compounded in each PCR amplification cycle, they can be sources of significant error. The same primer pair can have different PCR efficiencies when cDNAs derived from different sources are being used, although the same sequence is being amplified. This probably results from organic or metal ion contaminants present in the RNA preparation used to generate cDNA for PCR amplification. By monitoring PCR product formation through multiple rounds of PCR cycles, termed PCR cycle titration, one can assess the efficiency of amplification for a particular template. Using these cycle titration measurements, one can establish whether a particular RNA such as ~-actin is actually expressed at similar levels in two different RNA sources. If so, one could then compare the abundance of a second PCR template in units relative to ~-actin, provided t hat the efficiency of the second template was also equal in the two samples (50). T he efficiency of PCR can be determined by using radioactive PCR primers and quantifying the PCR product generated after differing cycle numbers. Plotting the log of the PCR product versus the cycle number will generate a straight line if measurements are taken during the exponential phase of the PCR reaction (Fig. 2). The slope of the line will be determined by the PC R efficiency. If the slopes are the same in a comparison of amplification of the same template in two different
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WILDE, KLINMAN,
samples, the efficiencies of the PC R reactions are the same. One final caveat to this technique is the underlying assumption that the reverse transcription reaction efficiency is the same for the RNA samples being compared. The use of internal RNA standards present in the cDNA synthesis and subsequent PCR reaction can overcome this problem, as discussed elsewhere (50). Because the parameters for PCR amplification t hat are chosen for any particular MAPPing experiment are unlikely to be optimal for all primer pairs t hat are present, it is especially important to remember t hat the intensity of bands for different cytokines does not directly correlate with absolute mRNA concentration, because the efficiency of amplification for each cytokine mRNA is different and is compounded with each cycle of amplification. Our recommendation for garnering useful and accurate information about cytokine expression by PCR would be to screen samples of interest by MAPPing. If intriguing differences are found in expression of a few genes, these differences should be investigated carefully by PCR cycle titration techniques as outlined above. T he possibility t hat interesting differences would be missed or subject to misinterpretation due to artifact would thus be minimized.
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_ IG. 2. Results of three experiments, A, B, and C, using the same set of primers. Experiments A and B resulted in cycle titrations with equal slopes, but different y intercepts. Therefore, these two experiments can be compared and show t h a t there is approximately a fivefold difference in the abundance of the cDNA being measured. Experiment C has a slope different from that of either of the other two experiments, and thus data from this cannot be used to estimate relative amounts of this cDNA. Experiments A and B can be compared quantitatively only if these two samples have superimposed lines from a reference such as fi-actin which resulted from the same initial input amount of RNA.
AND BAUER
CONCLUSION In conclusion, cytokine MAPPing (or MAPPing of any family of related gene products) can yield semiquantitative data concerning relative proportions of RNA species that are present in activated cells. Its principal asset is the ability to extract rapidly a large amount of information about simultaneous transcriptional events that are so numerous or that occur in such a rare population of cells that repetitive applications of single primer PCR would be prohibitive. The interpretation of MAPPing results is subject to the same types of precautions that apply to single primer PCR, and it is always advisable to seek alternative methods to verify MAPPing results. Nevertheless, MAPPing is a versatile addition to a growing number of PCR applications that can be used routinely to explore complex physiology in pathologic states.
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