Alacrity of cells engaged in the immune response.

A hypothesis is postulated and experimental findings are given that a special kind of cell heterogeneity exists that renders cells differentially sensitive to the epitopal stimulus with the consequence that only a portion of ligand-specific cells engages in the response. The differential sensitivity is based on epigenetically driven cell-to-cell variations in the expressed gene products, such that every cell becomes unique in its molecular profile, and will preserve its individuality in 'sensing' the boundary conditions of the response milieu. The readiness of cells to engage in the response will be termed alacrity. High-alacrity cells are ready to respond under given conditions because their molecular expression pattern - both in qualitative and in quantitative terms - matches the response milieu. This heterogeneity has little to do with the BCR and TCR specificity, that is, not all antigen-specific cells respond to a stimulus, and cells failing to respond do so because their overall molecular pattern is inadequate to the conditions of the response milieu. The corollary to this proposition is that whatever physiological conditions prevail, some ligand-specific cells will likely be ready to engage in the response, because their uniqueness makes them differentially reactive to external signals. Although the pool of cells available for any response is restricted under any given boundary condition, some idle cells are saved to be 'in reserve'. Experiments are described that are compatible with this proposition, and approaches are suggested to elucidate the mechanism of developing and maintaining alacrity. This paper is a contribution to the Centennial conference in honour of Niels Kaj Jerne, held in Lisbon November 2011.

Gene expression in IFN-gamma-activated murine macrophages

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Gene expression in IFN-gamma-activated murine macrophages.

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Proteomic analysis of rare molecular species of translated polypeptides from a mouse fetal thymus cDNA library.

Proteomic patterns from an ordered cDNA library of mouse fetal thymus origin of an overall complexity of 1536 clones are described. Patterns have been analyzed at 96, 12, or 8 clones in a mixture, or as individual clones. Clones yield in some instances a single spot, in other cases a complex cluster or family of spots is formed. The determination of the clonal address (a six-digit number, indicating the interception of three pooling dimensions) by inspection of pools of 8, 12 or 16 clones is a reliable approach; nevertheless a complete proof consists in retrieving the clone and then submitting to transcription, translation and proteomic analysis. The spot clusters are meaningful clone identifiers; cluster components (families of polypeptides) are characteristic of individual clones and are independent of clones coexisting (and being co-expressed) in a given pool. A 'cluster' originates from a single cloned message and might be due to post-translational modification (offered by the reticuleocyte machinery) or as a result of programmed degradation. Thirteen clones or families of clonal products are shown, and the heterogeneity of the 'appearance' of clones is documented. In about half, the assignment of a clonal polypeptide product to a storage well position has failed; this might be due to a variety of considerations elaborated herein. The arguments are presented, that analysis of abundant proteins of a cell (activated lymphocyte) comprises 'classical proteomics', but for the analysis of the rare molecular species of proteins, Poissonian approaches of replicable material have to be used.

Proteome, transcriptome and genome: top down or bottom up analysis?

Biological systems are comprised of protein components found at a wide variety of abundances from millions of molecules of a single species per cell to less than one copy per cell. Because of this wide range of concentrations, measurement or a full accounting of each system is presently unavailable. Conventional separation and analytical methods (two-dimensional gel electrophoresis and mass spectrometry) allow identification and quantitation of many of the most abundant gene products (top down methods); and the majority of gene products, which are found at low abundance, can be neither identified nor measured in complex mixtures at present. The gene products that are found at low levels can be characterized and their properties analyzed by preparing ordered gene libraries of limited complexity from mRNA. When such preparations are expressed in cell free systems and analyzed by two-dimensional gel electrophoresis, the features of the gene products are available for analysis. This 'bottom up' approach allows identification of gene product properties so that analytical procedures can be devised and applied to complex mixtures.

Global analysis of gene expression in cells of the immune system I. Analytical limitations in obtaining sequence information on polypeptides in two-dimensional gel spots.

We have outlined various aspects and limitations of the collective analysis of protein species of a cell (lymphocyte). We have indicated research directions that, in to our opinion, deserve more attention. We have evaluated mainly the approach used in our laboratory and we recognize that a bulk of important research on the interface of proteomics and genomics remains to be dealt with. It is of great value that we can proceed in our quest by trial and error. But as much as the human genome initiative was not implemented by trial and error, but by formulating new technological approaches, we hope that our approach can be incorporated in the mainstream of proteomics. We need several integrating research directions, some of which are outlined in this communication, namely the use of ordered cDNA libraries, cell-free expression systems, high density filter hybridization, identification of two-dimensional (2-D) gel spots in terms of their amino acid composition through biosynthetic labeling and identification of restriction sites in the corresponding coding sequences. In the accompanying paper the cDNA ordered library approach will be described in some detail.

Global analysis of gene expression in cells of the immune system II. Cell-free translation products and high-density filter hybridization data.

We have developed an experimental system for linking information on cell-free transcription and translation products from cDNA clones with data obtained from hybridization signals from complex probes. The work described in this paper consists of two distinct processes, one being the construction of a system of clonal addresses and the other the identification of expressed genes involved in the studied processes. We describe the use of this system to identify genes involved in thymus development. Complex probes from fetal thymuses (GD15, 17 and newborn) of Balb/c mice were used to identify genes, which are up- or downregulated during the process of differentiation. The full set of information is available in the Clone-base of the Basel Institute for Immunology and will be retrievable from the website of the collaborating laboratories.

Increased protein synthesis after T cell activation in presence of cyclosporin A.

BACKGROUND: The immunosuppressive drug, cyclosporin A (CsA), blocks immune responses by inhibiting the calcineurin-dependent dephosphorylation of the nuclear factor of activated T cells (NFAT). We have previously reported that T cells activated in presence of CsA exhibit particular properties. In our study, we have tested the hypothesis that T cells activated in presence of CsA display a differential pattern of gene expression. METHODS: T lymphocytes were activated in vitro by Concanavalin A with or without CsA. The cells were: (1) pulsed with 35S-methionine to label the newly synthesized proteins that in turn were revealed by 2D-gel electrophoresis; (2) analyzed by flow cytometry for activation markers expression; and (3) examined by gel electrophoresis for early tyrosine phosphorylation events. RESULTS: The proteomic patterns of T lymphocytes activated by Concanavalin A, with or without CsA, were compared. In keeping with the well-known effect of the immunosuppressor, many polypeptides were not found in its presence. Remarkably, several newly synthesized polypeptides were detected only when activation was carried out in presence of CsA. In addition, immunologically relevant proteins, such as CD44 and CD69, escape CsA-inhibitory action. Furthermore, CsA did not modify the early protein tyrosine phosphorylation events resulting from T cell triggering. CONCLUSIONS: The present data show that the effect of CsA on protein synthesis is more complex than anticipated. Signaling provided by T cell activation and the blockade of the calcineurin-dependent pathway by CsA results in an altered program of gene expression.

Proteomic analysis of T cell activation in the presence of cyclosporin A: immunosuppressor and activator removal induces de novo protein synthesis.

Cyclosporin A (CsA), a fungal metabolite used in organ transplantation, blocks the immune responses by interfering with early activation signals preventing the induction of the IL2 gene. We have previously reported that the removal of the immunosuppressor provokes the transcription of the IL2 encoding gene. We have now investigated whether the transcription and translation of other genes accompanies this process. Withdrawal of CsA and Concanavalin A (ConA) from cultures of murine T cells activated by ConA in the presence of CsA leads to substantial changes in the pattern of radio-labelled proteins. A large number of polypeptides were synthesised de novo. In addition, a set of polypeptides detected prior to immunosuppressor elimination was not anymore synthesised. Finally, besides these qualitative changes, quantitative differences in terms of increased or decreased polypeptide abundance were also observed. The results demonstrate that activation in the presence of CsA has programmed the T cells to transcribe and translate a large number of genes, without further reactivation, once the immunosuppressor and the activator were removed.

Proteome analysis of activated murine B-lymphocytes.

Proteins extracted from murine B-lymphocytes after in vitro stimulation by lipopolysaccharide were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Structural information on the protein entities from 153 spots was obtained. Since many of these spots occur as members of spot families, a smaller number --98 genes-- was found to be coding for the identified spots. The elucidated proteins belong to groups of functional categories; we found 26 enzymes, 36 regulatory proteins, 15 chaperones, 15 structural proteins, 4 immunoglobulins, 1 ribosomal and 1 histone protein. A comparison between expected and observed molecular masses yields a good correlation for the majority of the compared spot entities. This set of proteins now identified in the context of a lymphocyte 2-D gel pattern should advance further studies on lymphocyte functions.

Translational control: a general mechanism for gene regulation during T cell activation.

Distributional changes of individual mRNAs between free ribonucleoprotein particles (mRNP) and ribosome-bound transcripts are used to assess translational control. Simultaneous analysis of many mRNA species is required to estimate the overall contribution of translation to the regulation of gene expression. To this purpose, total cytoplasmic RNA was fractionated in sucrose step gradients and poly(A)+ RNA was prepared from mRNP and ribosome-bound fractions. Since direct, simultaneous analysis of a profusion of mRNAs is not feasible, distribution of their in vitro translation products was examined after separation in 2-dimensional gels, followed by computer-based analysis of autoradiographs. When this analysis was applied to antigenically stimulated T cells, 36% of in vitro translation products showed a greater than 10-fold increase in intensity, suggesting transcriptional activation of the corresponding mRNAs. In comparison, 7.9% of individual mRNAs (54 of 685 species) were translationally activated. They were redistributed from free mRNP to ribosome-associated fractions; 4.7% (32 species) were translationally repressed, as indicated by the opposite pattern. The differential recruitment of 12.6% of mRNA species demonstrates specificity and the general significance of translational control during T cell activation, which implies that translation may play a similar role in regulating gene expression in a variety of physiological processes.

Expressed immunoglobulin repertoire of LPS-stimulated splenocytes of unimmunized mice as studied by two-dimensional gel electrophoresis.

The repertoire of isolated immunoglobulin polypeptide chains synthesized by LPS-stimulated splenic B cells from unimmunized 6 weeks old mice was studied by two-dimensional gel electrophoresis. These B cells formed mainly mu heavy chains, while only a small amount of gamma chains was detected on two-dimensional electrophoregrams. The number and character of spots corresponding to each class and type of H and L chains were analyzed. Most of the detected 52 spots, which corresponded to L chains, were well resolved with clearly defined round boundaries. Six of them belonged to two isotypes of lambda chains and the rest to the kappa chain. About 25 clusters corresponded to mu chains. They had different appearance from those of L chains and their characteristic elliptic form with prolonged vertical axes indicated the presence of several H chain variants of slightly different length (due probably to the length variations of CDR3 and carbohydrate heterogeneity) in each cluster. The limited number of spots both of H and L chains is explained as being due to restrictions in the expressed repertoire of preimmune splenic B cells, which have no somatic mutations in the immunoglobulin genes. The concept of macrorepertoire (referring to the relatively small number of detected molecular species) and microrepertoire (describing the mutationally altered molecules) is introduced.

Analysis of a randomly assorted cDNA library from BW 5147 lymphoid cells. Frequency estimates based on computer aided concatenation matching of 2D gel polypeptide products.

A cDNA library was divided into 291 sectors of low complexity, 800-1000 recombinant phage plaques per sector. Aliquots of DNA from sectors prepared from high titer phage were subjected to in vitro transcription using T7 polymerase and thereafter RNA was translated in a cell-free rabbit reticulocyte system in the presence of [35S] methionine. The resulting polypeptides were separated by 2D gel electrophoresis. Radiofluorographs of the gels prepared from 10 sectors were subjected to scanning and image analysis. A multilevel spot matching procedure was employed allowing us to recognise spot occurrence in the 10 sectors. The number of detected polypeptide spots per sector varied between 147 and 325. Overall, 1082 different spots were counted totalling 1983 spots considering multiple entries. The distribution of the spots and the frequency distribution of the RNA population among the 10 sectors are shown. The highest detected frequency is 10(-2), while one half of the RNA molecular species are present at a frequency of 10(-3) or lower.

Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway.

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and beta-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.

Neutrophils as a source of putative restriction proteases: degradation of mammalian and yeast proteins monitored by two-dimensional gel electrophoresis.

We have compared the ability of intact neutrophils to degrade a complex substrate of proteins from mammalian and yeast origin. The substrate was obtained by biosynthetic labeling, and subsequent lysis of K562 cells (leukemic cell line) and of yeast culture. The mammalian substrate consisted of 619 and the yeast substrate of 185 different polypeptides, as visualized and represented on two-dimensional gel patterns. Upon incubation of the mammalian substrate with neutrophils, the bulk of spots disappeared so rapidly that after 240 min of incubation only 21 spots were detectable. Just one spot remained unaltered in its intensity throughout the whole period of incubation. About 440 spots reveal a t1/2 shorter than 8 min. Yeast substrate is represented by a smaller number of the starting polypeptides (185) from which 55 spots "survive" the neutrophil treatment. About 30 spots have a t1/2 shorter than 8 min. We conclude that neutrophils are equipped with a potent proteolytic apparatus, and this is capable of eliminating various proteins in a highly efficient manner. The system is much less effective in eliminating proteins from distant species, like yeast. Although the cells governing and regulating the immune system are clearly of lymphoid origin, it might well be that the preimmune task of eliminating self antigens in a manner as predicted in the restriction protease hypothesis is performed by neutrophils.

Restriction sites as identification tags for lymphocyte cDNAs.

A cDNA library was prepared from BW 5147 murine lymphoma cells in lambda ecc III phage and randomly partitioned into 291 sectors, each with 800-1000 recombinant phage plaques. One sector was chosen for further characterization in terms of sensitivity to restriction endonuclease cutting. Aliquots of DNA preparations from this sector were treated with XhoI, SmaI, NcoI, PvuII, PstI, HindIII, EcoRI, BamHI, and ApaLI before being used as templates in a cell-free expression system. The polypeptide products were separated by two-dimensional (2-D) gel electrophoresis and radiofluorographs of the gels were submitted to computer-aided image analysis. The matched patterns were inspected for the presence or absence of spots upon individual endonuclease treatments. Thereafter the results were integrated in a data matrix which served as a basis to construct "restriction tags" for all spots. These (restriction) tags are binary numbers termed "cut numbers" and are a representation of the set of recognition sequences which are (or are not) part of the coding sequence. From 493 sequences (visualized as 2-D gel spots), 12 were not cut by any of the nine enzymes, while 45 were cut by all of them. The percentages of sequences resistant to enzyme treatment ranged between 17% and 77% for NcoI and XhoI, respectively. The enzyme treatments led to the appearance of a certain portion of "new spots", probably products from truncated sequences. From 512 possible cut numbers, 136 were assigned to the 493 spots. Restriction tags are available to facilitate retrieval of cDNA clones from the (partitioned) cDNA library.

Caffeic acid phenethyl ester profoundly modifies protein synthesis profile in type 5 adenovirus-transformed cloned rat embryo fibroblast cells.

Caffeic acid phenethyl ester (CAPE), an active component of propolis from bee hives, exerts a plethora of biological changes in diverse systems. These include antimitogenic, anticarcinogenic, anti-inflammatory and immunomodulatory responses. CAPE directly induces programmed cell death (apoptosis) in type 5 adenovirus (Ad5)-transformed cloned rat embryo fibroblast cells, wt3A. To identify the gene and protein expression changes induced by CAPE in wt3A cells we used a strategy involving in vitro translation of mRNAs followed by high resolution two-dimensional (2D) gel electrophoresis. This approach results in the detection of 745 spots, including 172 displaying differences in expression upon exposure to CAPE. A high proportion of spots show profound changes in spot intensity (42 spots with increased and 27 spots with decreased intensity) following CAPE treatment. These studies provide a basis for comparing these changes to known protein patterns of various cell populations with an ultimate aim of identifying families of polypeptides responsible for the up- and down-regulation of cellular proteins during CAPE-induced apoptosis. Specific newly appearing or completely disappearing spots (52 and 51 molecular species, respectively) will be used to attempt to identify and retrieve their cDNA counterparts from an ordered cDNA library. These approaches represent a novel strategy for cloning genes associated with and potentially mediating apoptosis.