[Alternative activation of antigen-presenting cells: concepts and clinical relevance]. / Alternative Aktivierung antigenpräsentierender Zellen. Konzept und klinische Bedeutung.

Lymphocytes do not just act as immunological effector cells, but also play an important role in the regulation of the immune response. They are able to induce or suppress inflammatory reactions and this balancing function is reflected in the well-known Th1/Th2 concept. Lymphocytes depend on antigen presenting cells (APC) for induction of differentiation and specific activation mediated by antigen capture, processing and presentation. Thus, APC represent a link between innate and acquired immunity. In parallel to the Th1/Th2 dichotomy, APC may be subdivided into (a) pro-inflammatory, classically activated APC such as mature dendritic cells and IFN-gamma-activated effector macrophages, and (b) into anti-inflammatory, alternatively activated APC such as IL-10-activated immature dendritic cells and IL-4-induced suppressor macrophages. Alternatively activated APC may mediate induction and maintenance of tolerance towards allergens and environmental substances, control the course of inflammatory reactions, and participate in healing processes by enhancing angiogenesis. Malignant tumors and certain infectious agents may misuse alternatively activated APC for their purposes, thereby requiring counter-action by Th1 lymphocytes and classically activated APC. The concept of alternative activation thus confirms the important role of APC in maintaining the balance between induction and suppression of both inflammation and immunity and it opens new perspectives for the development of specific immunotherapeutic approaches.

Alternatively activated macrophages differentially express fibronectin and its splice variants and the extracellular matrix protein betaIG-H3.

Alternative activation of macrophages, induced by Th2 cytokines and glucocorticoids, is essential for the proper functioning of anti-inflammatory immune reactions. To this end, alternatively activated macrophages (aaMPhi) express a not yet fully unravelled set of genes including cytokines such as alternative macrophage activation-associated CC-chemokine (AMAC)-1 and pattern recognition molecules such as the scavenger receptor CD163. In order to further characterize the molecular repertoire of aaMPhi, differential gene expression was analyzed by combining subtractive suppression cloning and differential hybridization. We show here that aaMPhi induced by interleukin (IL)-4 overexpress the prototype extracellular matrix (ECM) protein fibronectin on the mRNA and protein level. This overall increase is accompanied by a shift in fibronectin splice variants from an embryonic to a mature pattern. In addition, the expression of another ECM protein, betaIG-H3, is also upregulated by IL-4 in aaMPhi. In contrast to IL-4 and in line with its inhibitory effect on wound healing, dexamethasone exerts a strongly suppressive effect on fibronectin and betaIG-H3 expression. In conclusion, overexpression of ECM proteins induced by IL-4 in macrophages suggests that aaMPhi may be involved in ECM deposition and tissue remodelling during the healing phase of acute inflammatory reactions and in chronic inflammatory diseases.

A highly thermostable endo-(1,4)-beta-mannanase from the marine bacterium Rhodothermus marinus.

Rhodothermus marimus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity. When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 degrees C and pH 5.4, respectively. Purified, E. coil-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 degrees C and 90 degrees C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 degrees C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan.

Pseudoexons and regulatory elements in the genomic sequence of the beta-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1.

Recently, the authors reported the cloning of a novel human CC chemokine of alternatively activated macrophages (AMAC-1), whose expression is induced by Th2-associated cytokines such as interleukin 4 (IL-4), IL-13 and IL-10; vice versa, AMAC-1 expression is inhibited by Th1-associated cytokines such as interferon gamma (IFN-gamma). In order to study the genomic organization and transcriptional regulation of the AMAC-1 gene, genomic clones were isolated by screening a human lambda genomic library. Sequencing of a clone with a 1.7-kb insert gave a partial genomic sequence for the AMAC-1 gene. The complete AMAC-1 genomic sequence was obtained by bioinformational methods and the whole region spanning the AMAC-1 gene was verified by PCR amplification of subfragments and sequencing. The AMAC-1 gene consists of three exons. Whereas exons 2 and 3 were separated by a small intron of 411 bp, exon 1 and exon 2 were separated by 6 kb of non-translated genomic sequence containing two pseudoexons that are not expressed although they feature intact exon/intron boundaries and complete open reading frames. In order to allow a detailed analysis, a 2.7-kb fragment containing the promoter region and the first exon of AMAC-1 gene was cloned into a reporter gene construct. In the AMAC-1 promoter, two possible transcription start points were identified. In addition, several putative regulatory sequences for IL-4- and IFN-gamma-dependent transcriptional pathways were found including STAT6 and STAT1 binding sites as well as several AP-1 and C/EBP elements. Interestingly, a combined STAT6/STAT1 binding element is located in the direct vicinity of the first putative transcription start point. Competitive binding of IL-4-induced STAT6 versus IFN-gamma-induced STAT1 to this site may explain the antagonistic effects these cytokines exert on AMAC-1 expression.

Alternative versus classical activation of macrophages.

In parallel to the Th1/Th2 paradigm, antigen-presenting cells (APC) are divided into classically activated APC (dendritic cells/effector macrophages) and alternatively activated APC (IL-4-induced, alternatively activated macrophages/IL-10-induced, immature dendritic cells). Alternatively activated APC share a special molecular repertoire including receptors of innate immunity with broad specificity for foreign antigen and anti-inflammatory cytokines such as IL-1Ra and alternative macrophage activation-associated CC-chemokine-1. Alternatively activated APC mediated tolerance and downregulated inflammation. Abuse of alternatively activated APC in support of infectious susceptibility or tumor immune escape is counteracted by the classical pathway. Thus, classically and alternatively activated APC secure the balance between proinflammatory and anti-inflammatory immune reactions.

Langerhans cells do not express alternative macrophage activation-associated CC chemokine (AMAC)-1.

We have cloned a novel human CC chemokine, alternative macrophage activation-associated CC chemokine (AMAC)-1 that is highly homologous to macrophage inflammatory protein (MIP)-1alpha. In contrast to MIP-1alpha, AMAC-1 is induced in macrophages by Th2-associated cytokines IL4, IL13, and IL10 in vitro; in addition, AMAC-1 is expressed by Th1-suppressive alveolar macrophages in vivo. Surprisingly, however, AMAC-1 is also expressed by GM-CSF-induced, in vitro monocyte-derived dendritic cells when treated by IL4. Here, we present a detailed analysis of AMAC-1 expression in monocyte-derived dendritic cells in vitro and show that the prime dendritic cells in vivo, i.e. epidermal Langerhans cells, do not express AMAC-1 mRNA. In conclusion, AMAC-1 is a novel CC chemokine whose Th2-associated expression pattern in alternatively activated suppressor macrophages in vivo and in vitro and its absence from epidermal Langerhans cells in vivo suggest that it may be involved in inhibition of Th1 reactions and in tolerance induction.

The laminarinase from thermophilic eubacterium Rhodothermus marinus--conformation, stability, and identification of active site carboxylic residues by site-directed mutagenesis.

A gene (lamR) encoding laminarinase (LamR) was cloned from the marine thermophilic eubacterium Rhodothermus marinus ITI278. The enzyme purified from recombinant Escherichia coli cells hydrolyses mixed 1,3-1,4-beta-glucans (lichenan, barley and oat beta-glucan) and 1,3-beta-homoglucans (laminarin, curdlan) by an endo type action pattern. The CD spectrum of laminarinase is characteristic for a protein with prevailing beta secondary-structural elements, and the fluorescence spectrum points to a surface localisation of the tryptophan residues. A half-transition concentration of 5.4 M guanidinium chloride was measured for the denaturant-induced unfolding of laminarinase monitoring changes of the ellipticity at 222 nm and the fluorescence. Substitution of acidic residues Glu129, Asp131 and Gln134, which are invariant in family 16 glycosyl hydrolases, caused a severe reduction of beta-glucan-hydrolysing activity suggesting their central role in enzymatic hydrolysis. Deletion of Met133 drastically reduced catalytic activity. Met133 is invariant in family 16 laminarinases but not present in the active-site region of bacterial 1,3-1,4-beta-glucanases which also belong to glycosyl hydrolase family 16. Replacement of Met133 by Ala, Cys or Trp did not affect activity against 1,3-1,4-beta-polyglucans and 1,3-beta-polyglucans, but in mutant Met133A the rate of hydrolysis of cellobiosyltriose (G1-4G1-3Gr) was increased about 10 times. Hydrolysis of 1,3-beta-oligosaccharides and 1,4-beta-oligosaccharides (DP 2-7) demonstrated the ability of the enzyme to cleave 1,3-beta-linkages and 1,4-beta-linkages in low-molecular-mass carbohydrates independent of the structure of neighbouring linkages. The laminarinase contains five or six subsites for substrate binding according to the action pattern deduced from hydrolysis of labelled and unlabelled curdlan oligosaccharides of different chain length.

Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern.

We have cloned a novel human CC-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1. The isolated cDNA clone (803 bp) shows a single open reading frame of 267-bp coding for 89 amino acid residues; mature AMAC-1 protein is predicted to consist of 69 amino acids with a m.w. of 7855. Sequence alignment and 3D-modeling show the typical structural characteristics of CC-chemokines with special features in the receptor-activating domain. AMAC-1 is most closely related to MIP-1 alpha with a cDNA and protein sequence homology of 55% and 59%, respectively. However, the expression pattern of AMAC-1 is directly opposite to that of MIP-1 alpha. While MIP-1 alpha is induced by classical macrophage mediators such as LPS and is inhibited by IL-4 and glucocorticoids, AMAC-1 is specifically induced in macrophages by alternative macrophage mediators such as IL-4, IL-13, and IL-10. Expression of AMAC-1 is inhibited by IFN-gamma while glucocorticoids exert a slightly positive synergistic effect in combination with IL-4. Peripheral blood monocytes do not express AMAC-1; time course experiments show that monocyte-to-macrophage differentiation is a prerequisite for AMAC-1 expression. Expression of AMAC-1 by granulocyte-macrophage CSF/IL-4-induced, monocyte-derived dendritic cells is complex; in mature adherent dendritic cells, however, only minor AMAC-1 mRNA expression was found. In vivo, AMAC-1 is expressed by alveolar macrophages from healthy persons, smokers, and asthmatic patients. In conclusion, AMAC-1 is a novel CC-chemokine whose expression is induced in alternatively activated macrophages by Th2-associated cytokines; thus, AMAC-1 may be involved in the APC-dependent T cell development in inflammatory and immune reactions.

Individual amino acids in the N-terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases.

Thermostability and unfolding behavior of the wild-type (1,3-1,4)-beta-glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12-M) delta F14 and H(A12-M) delta Y13F14A were studied by spectroscopic and microcalorimetric measurements. H(A12-M) delta F14 is constructed by the fusion of 12 N-terminal amino acids of AMY with amino acids 13-214 of MAC, and by deletion of F14. In H(A12-M) delta Y13F14A, the N-terminal region of MAC is exchanged against the AMY sequence, Y13 is deleted, and Phe 14 is exchanged against Ala. The sequence of the N-terminal loop region from Pro 9 to amino acid 16 (or 17) is very important for the properties of the enzymes and influences the effects of Ca2+ ions on the thermostability and unfolding behavior of the enzymes. The half transition temperatures T(m) are higher in the presence of Ca2+ than in Ca2+ free buffer. Furthermore, the unfolding mechanism is influenced by Ca2+. In Ca(2+)-free buffer, MAC, H(A12-M) delta F14 and H(A12-M) delta Y13F14A unfold in a single cooperative transition from the folded state to the unfolded state, whereas for AMY, a two-step unfolding was found. In the presence of Ca2+, the two-step unfolding of AMY is strengthened. Furthermore, for H(A12-M) delta F14, a two-step unfolding is induced by Ca2+. These data indicate a two-domain structure of AMY and H(A12-M) delta F14, in the presence of Ca2+. Thus, point mutations in a peripheral loop region are decisive for thermal stabilities and unfolding mechanisms of the studied glucanases in the presence of Ca2+.

Influence of Ca2+ on conformation and stability of three bacterial hybrid glucanases.

The three hybrid glucanases (1-12)AMY x MAC(13-214), (1-12)AMY x des-Tyr13MAC(14-214); (1-16)AMY x MAC(17-214) are composed of short N-terminal segments of 12 or 16 amino acid residues derived from the Bacillus amyloliquefaciens glucanase (AMY) and of residues 13-214, 14-214 and 17-214, respectively, derived from the Bacillus macerans enzyme (MAC). The three proteins have similar conformational features as shown by the similar characteristics of their CD spectra in the far- and near-ultraviolet region. A metal-ion-binding site was identified in the hybrid glucanase (1-16)AMY x MAC(17-214) by a crystal structure analysis [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291]. Only minor conformational changes of the three hybrid glucanases were observed depending on the presence or absence of Ca2+ ions but for (1-16)AMY x MAC(17-214) and (1-12)AMY x des-Tyr13MAC(14-214) the occupation of this metal-binding site by a Ca2+ ion is connected with a large increase of the stability against thermal and chemical unfolding. Surprisingly, for (1-12)AMY x MAC(13-214), which differs from (1-12)AMY x des-Tyr13MAC(14-214) by only one additional amino acid in an N-terminal loop region, the effect of Ca2+ ions on the stability is small. The exchange of a few amino acid residues near the N-terminus of the B. macerans glucanase against amino acids found at comparable positions in the B. amyloliquefaciens glucanase seems to influence very strongly the strength of the Ca2+ binding site and concomitantly the stability of the hybrid glucanases.

Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase.

In beta-glucans those beta-1,4 glycosidic bonds which are adjacent to beta-1,3 bonds are cleaved by endo-1,3-1,4-beta-glucanases (beta-glucanases). Here, the relationship between structure and activity of the beta-glucanase of Bacillus macerans is studied by x-ray crystallography and site-directed mutagenesis of active site residues. Crystal structure analysis at 2.3-A resolution reveals a jelly-roll protein structure with a deep active site channel harboring the amino acid residues Trp101, Glu103, Asp105, and Glu107 as in the hybrid Bacillus beta-glucanase H(A16-M) (Keitel, T., Simon, O., Borriss, R., and Heinemann, U. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5287-5291). Different mutant proteins with substitutions in these residues are generated by site-directed mutagenesis, isolated, and characterized. Compared with the wild-type enzyme their activity is reduced to less than 1%. Several mutants with isosteric substitutions in Glu103 and Glu107 are completely inactive, suggesting a direct role of these residues in glycosyl bond hydrolysis. The kinetic properties of mutant beta-glucanases and the crystal structure of the wild-type enzyme are consistent with a mechanism where Glu103 and Glu107 are the catalytic amino acid residues responsible for cleavage of the beta-1,4 glycosidic bond within the substrate molecule.

Microcalorimetric determination of the thermostability of three hybrid (1-3,1-4)-beta-glucanases.

Thermodynamic parameters of the three hybrid (1-3,1-4)-beta-glucanases H(A12-M), H(A12-M) delta Y13, and H(A16-M) composed of short N-terminal regions derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the homologous Bacillus macerans enzyme were determined in 2 mM sodium cacodylate pH 6.0, 1.5M guanidine hydrochloride, containing 1 mM CaCl2 or 1 mM EDTA. Melting of H(A12-M) delta Y13 and H(A16-M) in the presence of calcium ions is characterized by two subtransitions; only one transition is observed in the case of H(A12-M). In calcium-free buffer each of the three hybrid enzymes melts in one two-state transition. Transition temperatures Tm and molar enthalpy changes delta H are reduced in the absence of calcium ions but the reduction is much more pronounced for H(A12-M) delta Y13 and H(A16-M) than for the less thermostable enzyme H(A12-M).

Determinants for the enhanced thermostability of hybrid (1-3,1-4)-beta-glucanases.

Hybrid (1-3,1-4)-beta-glucanases which contain an N-terminal region derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the closely related B. macerans enzyme may exhibit a thermostability superior to both parental enzymes. A systematic series of hybrid enzymes were constructed in order to delineate the amino acid residues that affect protein stability. Hybrid enzymes with between one and four of the N-terminal residues for the mature B. amyloliquefaciens (1-3,1-4)-beta-glucanase exhibit no significant changes in biochemical characteristics as compared with the parental B. macerans enzyme. However, significantly enhanced thermostability was observed in the hybrid enzyme containing an N-terminal segment of eight amino acid residues derived from the B. amyloliquefaciens enzyme. Site-directed mutagenesis revealed that the combined effect of Gln1, Thr2, Ser5 and Phe7 confer enhanced stability on hybrid enzymes, probably by improving the hydrogen bonding that stabilizes the interactions between the N-terminal and the centre of the folded molecule, as well as between the two termini of the polypeptide chain. Furthermore, deletion of Tyr13 in the hybrid enzyme containing the 12 N-terminal amino acids from the B. amyloliquefaciens (1-3,1-4)-beta-glucanase results in a dramatic increase in stability at 70 degrees C with the half-life of 6 min increased to around 4 h. This is twofold higher than the hitherto most stable hybrid enzyme in which the N-terminal domain consisted of 16 residues of the B. amyloliquefaciens enzyme.