Reduced proportions of natural killer T cells are present in the relatives of lupus patients and are associated with autoimmunity

Introduction

Systemic lupus erythematosus is a genetically complex disease. Currently, the precise allelic polymorphisms associated with this condition remain largely unidentified. In part this reflects the fact that multiple genes, each having a relatively minor effect, act in concert to produce disease. Given this complexity, analysis of subclinical phenotypes may aid in the identification of susceptibility alleles. Here, we used flow cytometry to investigate whether some of the immune abnormalities that are seen in the peripheral blood lymphocyte population of lupus patients are seen in their first-degree relatives.

Methods

Peripheral blood mononuclear cells were isolated from the subjects, stained with fluorochrome-conjugated monoclonal antibodies to identify various cellular subsets, and analyzed by flow cytometry.

Results

We found reduced proportions of natural killer (NK)T cells among 367 first-degree relatives of lupus patients as compared with 102 control individuals. There were also slightly increased proportions of memory B and T cells, suggesting increased chronic low-grade activation of the immune system in first-degree relatives. However, only the deficiency of NKT cells was associated with a positive anti-nuclear antibody test and clinical autoimmune disease in family members. There was a significant association between mean parental, sibling, and proband values for the proportion of NKT cells, suggesting that this is a heritable trait.

Conclusions

The findings suggest that analysis of cellular phenotypes may enhance the ability to detect subclinical lupus and that genetically determined altered immunoregulation by NKT cells predisposes first-degree relatives of lupus patients to the development of autoimmunity.     

A European focus on proteomics

A report on the First International Symposium of the Austrian Proteomics Platform, Seefeld, Austria, 26-29 January 2004.     

MEK5, a new target of the atypical protein kinase C isoforms in mitogenic signaling

The MEK5-extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. Here we demonstrate that the aPKCs interact in an EGF-inducible manner with MEK5 and that this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, we show that zetaPKC and lambda/iotaPKC activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, we conclude that MEK5 is a critical target of the aPKCs during mitogenic signaling.     

Activation of IkappaB kinase beta by protein kinase C isoforms

The atypical protein kinase C (PKC) isotypes (lambda/iotaPKC and zetaPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha) and are required for the activation of NF-kappaB by this cytokine through a mechanism that most probably involves the phosphorylation of IkappaB. The inability of these PKC isotypes to directly phosphorylate IkappaB led to the hypothesis that zetaPKC may use a putative IkappaB kinase to functionally inactivate IkappaB. Recently several groups have molecularly characterized and cloned two IkappaB kinases (IKKalpha and IKKbeta) which phosphorylate the residues in the IkappaB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-kappaB through the activation of the IKKs. We report here that alphaPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zetaPKC positively modulates IKKbeta activity but not that of IKKalpha, whereas the transfection of a zetaPKC dominant negative mutant severely impairs the activation of IKKbeta but not IKKalpha in TNF-alpha-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKbeta, which is entirely dependent on the activity of alphaPKC but not that of the atypical isoforms. In contrast, the inhibition of alphaPKC does not affect the activation of IKKbeta by TNF-alpha. Interestingly, recombinant active zetaPKC and alphaPKC are able to stimulate in vitro the activity of IKKbeta but not that of IKKalpha. In addition, evidence is presented here that recombinant zetaPKC directly phosphorylates IKKbeta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappaB pathway at the level of IKKbeta activation and IkappaB degradation.     

The interaction of p62 with RIP links the atypical PKCs to NF-kappaB activation.

The two members of the atypical protein kinase C (aPKC) subfamily of isozymes (zetaPKC and lambda/iotaPKC) are involved in the control of nuclear factor kappaB (NF-kappaB) through IKKbeta activation. Here we show that the previously described aPKC-binding protein, p62, selectively interacts with RIP but not with TRAF2 in vitro and in vivo. p62 bridges the aPKCs to RIP, whereas the aPKCs link IKKbeta to p62. In this way, a signaling cascade of interactions is established from the TNF-R1 involving TRADD/RIP/p62/aPKCs/IKKbeta. These observations define a novel pathway for the activation of NF-kappaB involving the aPKCs and p62. Consistent with this model, the expression of a dominant-negative mutant lambda/iotaPKC impairs RIP-stimulated NF-kappaB activation. In addition, the expression of either an N-terminal aPKC-binding domain of p62, or its C-terminal RIP-binding region are sufficient to block NF-kappaB activation. Furthermore, transfection of an antisense construct of p62 severely abrogates NF-kappaB activation. Together, these results demonstrate that the interaction of p62 with RIP serves to link the atypical PKCs to the activation of NF-kappaB by the TNFalpha signaling pathway.     

Protein kinase C-independent expression of stromelysin by platelet-derived growth factor, ras oncogene, and phosphatidylcholine-hydrolyzing phospholipase C.

Changes in the expression of several genes play critical roles in cell growth and tumor transformation. A number of proteases are increased in some tumors, and the level of these enzymes correlates with the metastatic potential of several cancer cell lines. Stromelysin, with the widest substrate specificity, can degrade the extracellular matrix conferring metastatic potential to tumor cells. The mechanisms whereby growth factors and oncogenes control the expression of stromelysin are beginning to be characterized. In the study shown here we also identify a region in the stromelysin promoter which is involved in the induction of stromelysin in response to platelet-derived growth factor, phosphatidylcholine-hydrolyzing phospholipase C, and ras oncogene. Our results are consistent with the notion that platelet-derived growth factor/phosphatidylcholine-hydrolyzing phospholipase C induces stromelysin gene expression through a phorbol myristate acetate/protein kinase C-independent mechanism by acting through elements in the stromelysin promoter distinct from the 12-O-tetradecanoylphorbol-13-acetate-responsive element.