Julie Milland

The Acute Phase Response in the Rodenta

In the rodent, the general response to acute inflammation and tissue damage is characterized by a complex rearrangement in the pattern of concentrations of proteins in the plasma leading to an increase in the sedimentation rate of erythrocytes, an increase in leukocyte concentration in the bloodstream, and a decrease in the hematocrit. Body temperature changes only slightly or not at all. The reasons for the change in plasma concentrations of proteins are changes in their rates of synthesis in the liver. Degradation of plasma proteins is not affected. The details of the acute phase response evolved in the interaction of species with their environment. Therefore, it is not surprising to find differences in the details of the acute phase response among species. For example, alpha 2-macroglobulin is a strongly positive acute phase reactant in the rat, but not in the mouse; C-reactive protein is a strongly positive acute phase protein in the mouse, but is not found in the rat. An inducible acute phase cysteine proteinase inhibitor system, which has evolved from a primordial kininogen gene, has been observed so far only in the rat. The changes in the synthesis rates of acute phase proteins during inflammation are closely reflected by corresponding changes in intracellular mRNA levels. In the liver, the capacity to induce the acute phase pattern of synthesis and secretion of plasma proteins probably develops around birth. Changes in mRNA levels are brought about by changes in transcription rates or by changes in mRNA stability. Kinetics of mRNA changes during the acute phase response differ for individual proteins. The main signal compound for eliciting the acute phase response in liver seems to be interleukin-6/interferon-beta 2/hepatocyte stimulating factor, whereas interleukin-1 leads to typical acute phase changes in mRNA levels only for alpha 1-acid glycoprotein, albumin, and transthyretin. Plasma protein genes are expressed in various extrahepatic tissues, such as the choroid plexus, the yolk sac, the placenta, the seminal vesicles, and other sites. All these tissues are involved in maintaining protein homeostasis in associated extracellular compartments by synthesis and secretion of proteins. Synthesis and secretion of plasma proteins in paracompartmental organs other than the liver is not influenced by the acute phase stimuli.

The Molecular Basis for Galα(1,3)Gal Expression in Animals with a Deletion of the α1,3Galactosyltransferase Gene

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes α1,3galactosyltransferase (α1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galα(1,3)Gal carbohydrate epitope was not observed as Galα(1,3)Gal staining was still present in tissues from GGTA1−/− animals. This shows that, contrary to previous dogma, α1,3GT is not the only enzyme able to synthesize Galα(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1−/− mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galα(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galα(1,3)Gal epitope. Galα(1,3)Gal synthesized by iGb3S, in contrast to α1,3GT, was resistant to down-regulation by competition with α1,2fucosyltransferase. Moreover, Galα(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 −/− mice. Galα(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.

Humans lack iGb3 due to the absence of functional iGb3-synthase: implications for NKT cell development and transplantation.

The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galα(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galα(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.

Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons

Abstract:  Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement‐mediated rejection, as shown here using non‐immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high‐level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement‐mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti‐Galα(1,3)Gal epitope (anti‐GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre‐sensitized with antibody were highly resistant to human complement‐mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non‐immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at ∼24‐h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti‐GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced ∼8‐fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.

α1,3-Galactosyltransferase knockout pigs are available for xenotransplantation: Are glycosyltransferases still relevant?

In the early 1990s, the Galα(1,3)Gal carbohydrate linkage was found to be the major xenoepitope causing hyperacute rejection. This carbohydrate, the antibodies that bind to it, and the enzyme that produces it (α1,3‐galactosyltransferase) were the foci of research by many groups. Nearly a decade later, α1,3‐galactosyltransferase knockout pigs were finally produced; hyperacute rejection could be avoided in these pigs. Having achieved this goal, enthusiasm declined for the study of glycosyltransferases and their carbohydrate products. To examine whether this decline was premature, we evaluate whether gene deletion has indeed solved the initial rejection problem or, in fact, created new problems. This review addresses this by examining the impact of the gene deletion on cell surface carbohydrate. Surprisingly, Galα(1,3)Gal is still present in α1,3‐galactosyltransferase knockout animals: it is possibly synthesized on lipid by iGb3 synthase. Furthermore, removal of αGal resulted in the exposure of the N‐acetyllactosamine epitope. This exposed epitope can bind natural antibodies and perhaps should be capped by transgenic expression of another transferase. We believe the continued study of glycosyltransferases is essential to examine the new issues raised by the deletion of α1,3‐galactosyltransferase.

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for golgi localization.

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of α1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the α1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of α1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser5. Mutagenesis of Ser5 to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of α1,2-fucosyltransferase.

Carbohydrate residues downstream of the terminal Galα(1,3)Gal epitope modulate the specificity of xenoreactive antibodies

Carbohydrates are involved in many immunological responses including the rejection of incompatible blood, tissues and organs. Carbohydrate antigens with Galα(1,3)Gal epitopes are recognized by natural antibodies in humans and pose a major barrier for pig‐to‐human xenotransplantation. Genetically modified pigs have been established that have no functional α1,3‐galactosyltransferase (α1,3GT), which transfers αGal to N‐acetyllactosamine (LacNAc) type oligosaccharides. However, a low level of Galα(1,3)Gal is still expressed in α1,3GT knockout animals in the form of a lipid, isoglobotrihexosylceramide (iGb3), which is produced by iGb3 synthase on lactose (Lac) type core structures. Here, we define the reactivity of a series of monoclonal antibodies (mAb) generated in α1,3GT−/− mice immunized with rabbit red blood cells (RbRBC), as a rich source of lipid‐linked antigens. Interestingly, one mAb (15.101) binds weakly to synthetic and cell surface‐expressed Galα(1,3)Gal on LacNAc, but strongly to versions of the antigen on Lac cores, including iGb3. Three‐dimensional models suggest that the terminal α‐linked Gal binds tightly into the antibody‐binding cavity. Furthermore, antibody interactions were predicted with the second and third monosaccharide units. Collectively, our findings suggest that although the terminal carbohydrate residues confer most of the binding affinity, the fine specificity is determined by subsequent residues in the oligosaccharide.

Interactions between the ectodomains of haemagglutinin and CD46 as a primary step in measles virus entry

Recombinant soluble forms of the ectodomains of measles virus haemagglutinin (sH) and of its receptor CD46 (sCD46) were obtained as a purified disulphide-bonded sH homodimer with an apparent molecular mass of 160 kDa and a purified sCD46 monomer with an apparent molecular mass of 60 kDa, without detectable contamination with moesin. Purified sH bound to purified and immobilized sCD46 and this binding was specifically inhibited by sCD46 in solution. sCD46 bound to wild-type H expressed on the cell surface and inhibited measles virus binding to CD46-expressing cells. Binding of sCD46 to cell surface H was increased about twofold when measles virus fusion protein was coexpressed with H. sH bound to wild-type cell surface CD46 and inhibited measles virus binding onto CD46-expressing cells. sCD46 also inhibited virus infection. Thus, the direct interaction between the ectodomains of H and CD46 is likely to be the primary event in measles virus infection.

The Cytoplasmic Tail of α1,3-Galactosyltransferase Inhibits Golgi Localization of the Full-length Enzyme

It is currently under debate whether the mechanism of Golgi retention of different glycosyltransferases is determined by sequences in the transmembrane, luminal, or cytoplasmic domains or a combination of these domains. We have shown that the cytoplasmic domains of α1,3-galactosyltransferase (GT) and α1,2-fucosyltransferase (FT) are involved in Golgi localization. Here we show that the cytoplasmic tails of GT and FT are sufficient to confer specific Golgi localization. Further, we show that the expression of only the cytoplasmic tail of GT can lead to displacement or inhibition of binding of the whole transferase and that cells expressing the cytoplasmic tail of GT were not able to express full-length GT or its product, Galα1,3Gal. Thus, the presence of the cytoplasmic tail prevented the localization and function of full-length GT, suggesting a possible specific Golgi binding site for GT. The effect was not altered by the inclusion of the transmembrane domain. Although the transmembrane domain may act as an anchor, these data show that, for GT, only the cytoplasmic tail is involved in specific localization to the Golgi.

Recognition of a carbohydrate xenoepitope by human NKRP1A (CD161)

Abstract:  Background:  Many immunologically important interactions are mediated by leukocyte recognition of carbohydrates via cell surface receptors. Uncharacterized receptors on human natural killer (NK) cells interact with ligands containing the terminal Galα(1,3)Gal xenoepitope. The aim of this work was to isolate and characterize carbohydrate binding proteins from NK cells that bind αGal or other potential xenoepitopes, such as N‐acetyllactosamine (NAcLac), created by the deletion of α1,3galactosyltransferase (GT) in animals.