Yael G. Alevy

Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease.

To understand the pathogenesis of chronic inflammatory disease, we analyzed an experimental mouse model of chronic lung disease with pathology that resembles asthma and chronic obstructive pulmonary disease (COPD) in humans. In this model, chronic lung disease develops after an infection with a common type of respiratory virus is cleared to only trace levels of noninfectious virus. Chronic inflammatory disease is generally thought to depend on an altered adaptive immune response. However, here we find that this type of disease arises independently of an adaptive immune response and is driven instead by interleukin-13 produced by macrophages that have been stimulated by CD1d-dependent T cell receptor–invariant natural killer T (NKT) cells. This innate immune axis is also activated in the lungs of humans with chronic airway disease due to asthma or COPD. These findings provide new insight into the pathogenesis of chronic inflammatory disease with the discovery that the transition from respiratory viral infection into chronic lung disease requires persistent activation of a previously undescribed NKT cell–macrophage innate immune axis.

Long-term IL-33–producing epithelial progenitor cells in chronic obstructive lung disease

Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus infection in mice revealed an unexpected role for innate immune cells in IL-13-dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD). In the mouse model, IL-33/IL-33 receptor signaling was required for Il13 and mucin gene expression, and Il33 gene expression was localized to a virus-induced subset of airway serous cells and a constitutive subset of alveolar type 2 cells that are both linked conventionally to progenitor function. In humans with COPD, IL33 gene expression was also associated with IL13 and mucin gene expression, and IL33 induction was traceable to a subset of airway basal cells with increased capacities for pluripotency and ATP-regulated release of IL-33. Together, these findings provide a paradigm for the role of the innate immune system in chronic disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production.

IL-13–induced airway mucus production is attenuated by MAPK13 inhibition

Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics. Here, we define a signaling pathway from chloride channel calcium-activated 1 (CLCA1) to MAPK13 that is responsible for IL-13-driven mucus production in human airway epithelial cells. The same pathway was also highly activated in the lungs of humans with excess mucus production due to COPD. We further validated the pathway by using structure-based drug design to develop a series of novel MAPK13 inhibitors with nanomolar potency that effectively reduced mucus production in human airway epithelial cells. These results uncover and validate a new pathway for regulating mucus production as well as a corresponding therapeutic approach to mucus overproduction in inflammatory airway diseases.

Peripheral blood microchimerism in human liver and renal transplant recipients : Rejection despite donor-specific chimerism

BACKGROUND Development of donor-specific microchimerism (DSM) has been proposed as one of the possible mechanisms for induction and maintenance of allograft tolerance. The aim of this study was to determine: (1) the state of DSM in liver transplant (LTx) and renal transplant (RTx) recipients, (2) whether the persistent presence of an allograft is a requirement for maintenance of chimerism, and (3) whether donor-specific blood transfusions (DST) facilitate chimerism development in RTx recipients and whether this correlates with allograft function. METHODS Qualitative and quantitative analysis of DSM in peripheral blood of LTx and RTx recipients was assessed by polymerase chain reaction and competitive polymerase chain reaction using HLA-DR probes for mismatched antigens between the donor and recipient. RESULTS LTx recipients (11 of 12) who had or were having rejection were positive for DSM in circulation compared with 4 of 11 with normal allograft function (P<0.01). The number of donor cells did not correlate with allograft function. LTx recipients (4 of 4) who lost their first allograft and underwent retransplantation retained DSM for the first donors. RTx recipients who received DST (8 of 8) were positive for DSM compared with 6 of 12 of nontransfused recipients (P<0.045). CONCLUSIONS The results suggest that LTx and RTx recipients undergo rejection despite DSM. The development of DSM may not be a prerequisite for normal allograft function. Once DSM is established, the presence of the allograft is not required for maintenance of chimerism. DST facilitated the development of DSM in RTx recipients. Direct correlation was not observed between the development of DSM and allograft function in either DST or nontransfused RTx recipients.

Characterization of HDJ-2, a human 40 kD heat shock protein

Heat shock proteins (HSP) are a large and complex family of proteins that play important roles in cellular function and survival. In previous studies, cDNA for a 45 kD human HSP (HDJ-2) was cloned and shown to be homologous to DNA-J, a bacterial HSP [F.M. Ausubel, R. Brent, R. E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1997; A. Chellaiah, A. Davis, T. Mohanakumar, Cloning of a unique human homologue of the Escherichia coli DNAJ heat shock protein, Biochim. Biophys. Acta 1174 (1993) 111-113]. We have also shown that the expression of HDJ-2 is highly elevated in kidney allograft biopsies of kidneys undergoing rejection [Y.G. Alevy, D. Brennan, S. Durriya, T. Howard, T. Mohanakumar, Increased expression of the HDJ-2 heat shock protein in biopsies of human rejected kidneys, Transplantation 61 (1996) 963-967]. Because of the potential importance of HDJ-2 to disease pathogenesis, we carried out studies to characterize the structure and regulation of HDJ-2. Polyclonal and monoclonal antibodies that recognize recombinant HDJ-2 were prepared and used to localize its cellular expression. HDJ-2 was found to be farnesylated but not glycosylated. This HSP was ubiquitously expressed in all of the cell types we analyzed and was localized throughout the cytoplasm and around the nuclear membrane. However, upon heat shock it migrated to the Golgi, nucleolus, and the nuclear membrane. Northern blot analysis revealed two mRNA transcripts whose synthesis was not affected by heat shock. In addition, Western blot analysis showed that expression of HDJ-2 was also not affected by heat shock. Thus, our study shows the characterization of a HSP which, because of its migration pattern upon heat shock, is an excellent candidate for a protein chaperon.

IL13 activates autophagy to regulate secretion in airway epithelial cells

ABSTRACT Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation

Background: CLCA proteins activate CaCCs; CLCAs have roles in cancer and inflammatory lung diseases, but their mechanism of action is unknown. Results: CLCA proteins must undergo self-cleavage via their own novel metalloprotease domain in the N terminus to activate CaCCs. Conclusion: Self-cleavage unmasks the N-terminal fragment, which alone activates CaCCs. Significance: This work identifies a unique ion channel activation mechanism defining framework to understand CLCA functions in diseases. The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

Increased expression of the HDJ-2 heat shock protein in biopsies of human rejected kidney

The presence of donor-specific alloreactive helper and cytotoxic T cells has been described in allograft biopsies obtained from individuals undergoing acute allograft rejection of various solid organs. However, not all of these lymphocytes demonstrated specificity to mismatched donor HLA antigens. The identity of the antigens to which these T cells are directed to is still unknown at present. The possibility that heat shock proteins (Hsp) could serve as antigenic determinants to which these T cells respond has been raised. We have recently cloned and characterized a novel Hsp of 45Kd molecular weight. In the present study we show that the synthesis of this Hsp (HDJ-2) as well as Hsp60 is significantly elevated in kidney biopsies from individuals undergoing acute and chronic rejection. No message was detected either for HDJ-2 or Hsp60 in biopsies obtained from normal pretransplant kidneys or posttransplant kidneys with no rejection. However, there was some increase in Hsp in miscellaneous causes of allograft dysfunction such as infection and drug allergy. But, this was not as consistent as that noted for allograft rejection. This marked increase in Hsp expression during allograft rejection suggests Hsps as potential candidates for antigenic determinants contributing to kidney rejection.

Evidence that pediatric liver transplant recipients may undergo late rejection episodes in spite of donor-specific microchimerism

Lymphocytes of donor origin can be demonstrated in the blood of many liver transplant recipients. It has been proposed that this chimerism may imply graft tolerance and permit withdrawal of immunosuppression. We report two children with liver transplants who had lymphocyte chimerism demonstrated at the time of late rejection episodes. One child was chimeric for both of his donors, although he retained the first allograft for only 3 days. Thus, the persistence of donor lymphocytes may be unrelated to the presence of the donor organ. Graft rejection can occur in spite of donor-specific microchimerism. The role of donor-specific microchimerism in graft acceptance or graft tolerance remains to be elucidated.

CD32A (FcγRIIa) mRNA expression and regulation in blood monocytes and cell lines

Abstract The cell surface expression of the CD32 receptors for the Fc portion of immunoglobulin G (Fc γ RIIa) is highly regulated by agents such as phorbol ester (PMA) and cytokines. In this study we investigated the regulatory effects of PMA, aggregated IgG (AIgG) and KuFc79 anti-CD32 monoclonal antibodies (mAb) on the expression of the CD32A isomer mRNA. When U937 (CD32 + cells) are incubated with PMA a significant enhancement of the CD32A isomer mRNA is observed. The CD32A mRNA is also markedly enhanced when the CD32 + K562 cells are incubated with AIgG and anti-CD32 mAb but not with control MOPC-195 mAb. The addition of actinomycin D (ActD), a transcriptional inhibitor together with PMA, AIgG or KuFc79 mAb diminishes the enhanced levels of CD32A mRNA to the basal, constitutively expressed levels, implicating transcriptional regulation in this modulatory process. The PMA induced mRNA is rapidly degraded while the constitutively expressed CD32A mRNA is not, suggesting differential regulation of the stimulated vs the unstimulated CD32A mRNA. Inhibition of protein synthesis does not significantly affect the upregulation of CD32 mRNA by PMA, AIgG or KuFc79 in U937 and K562 cells. The upregulation of CD32A mRNA observed in the cell lines U937 and K562 is also detected when normal blood monocytes are used. Similarly to the cell lines the enhancement of CD32A mRNA in monocytes is blocked by ActD.