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.

Increased expression of HDJ-2 (heat shock protein 40) and heat shock protein 70 in biopsy specimens of transplanted human lungs.

BACKGROUND: Heat shock proteins are expressed during several forms of stress and inflammation. This study was done to determine whether the expression of heat shock protein HDJ-2 (heat shock protein 40), heat shock protein 60, and heat shock protein 70 are increased during rejection in human pulmonary allografts. METHODS: Thirty-five transbronchial biopsy specimens were obtained from adult lung transplant recipients. Histologic analysis and assessment of heat shock protein HDJ-2, heat shock protein 60, and heat shock protein 70 mRNA expression was performed. Total RNA was extracted, reverse transcribed, and amplified by polymerase chain reaction with oligonucleotide primers specific for the heat shock proteins. The identity of the amplified message was verified by Southern blot and slot blot analysis. RESULTS: The expression of heat shock protein HDJ-2 was significantly higher in samples from lung transplant recipients undergoing rejection when compared with recipients without rejection or infection. Heat shock protein 70 expression was also increased in rejection. Expression of heat shock protein 60 did not show any increase in recipients with no evidence of rejection and infection or transplant recipients with rejection or infection. Serial analysis of heat shock protein HDJ-2 and heat shock protein 70 obtained in biopsy specimens during and after rejection showed a decrease of heat shock protein HDJ-2 and heat shock protein 70 expression after resolution of lung rejection. CONCLUSION: Our data demonstrate that the expression of heat shock protein HDJ-2 and heat shock protein 70 increases during lung rejection. However, only heat shock protein HDJ-2 was able to differentiate between rejection and infection. Measurement of heat shock protein HDJ-2 in transbronchial biopsy specimens may assist in the differential diagnosis between rejection and infection in lung transplant recipients.

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.

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.

Cytokine gene transcripts for tumor necrosis factor-alpha, interleukin-2, and interferon-gamma in human pulmonary allografts.

BACKGROUND: Cytokines participate in host responses to allografts, largely through recruiting and activating various regulatory and effector cells. We performed this study to determine the feasibility of using polymerase chain reaction methodology to define the expression of three important cytokines (tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) in human pulmonary allografts. METHODS: Twenty-six graft-derived samples (11 transbronchial biopsy and 8 macrophage and 7 lymphocyte cell pellets isolated from bronchoalveolar lavage) were obtained from 13 lung transplant recipients and treated as follows: extraction of RNA; reverse transcription of RNA to complementary DNA; polymerase chain reaction amplification of cDNA with oligonucleotide primers specific for the three cytokines; gel electrophoresis of the polymerase chain reaction products; and verification of correct cytokine message by Dot blot technique (with specific 32P-labeled oligonucleotide probes). RESULTS: Concomitant pathologic evaluation of biopsy specimens from these 13 recipients showed five diagnostic groups: "normal" (no rejection/infection), n = 2; acute rejection, n = 4; nonspecific inflammation, n = 3; infection, n = 3; and obliterative bronchiolitis, n = 1. Interleukin-2 was expressed predominantly in acute rejection and infection (seven of ten and five of six samples positive, respectively), whereas tumor necrosis factor-alpha was expressed mainly in nonspecific inflammation (four of five samples) and somewhat less in rejection (six of ten). Interferon-gamma was expressed less frequently (in two of six samples with infection, but in none of ten with rejection and none of five with nonspecific inflammation). Serial data from one patient (6 months apart) showed considerable increase in interleukin-2 and interferon-gamma expression as she progressed from normal histologic status to obliterative bronchiolitis. CONCLUSIONS: Cytokine gene transcripts can be determined from minute samples derived directly from pulmonary allografts. Although our data are insufficient to make definitive conclusions, the suggestion of trends of cytokine expression in different posttransplantation pathologic conditions may indicate a useful role for this approach in the clinical evaluation of the lung transplant recipient.

CD32C (Fc gamma RIIC) mRNA expression and regulation.

The cell surface expression of the CD32 receptors for the Fc portion of immunoglobulin G (Fc gamma RII-CD32) is regulated by agents such as phorbol esters (PMA) and cytokines. In this study, we investigated the effects of PMA and interferon-gamma (IFN-gamma) on the expression of CD32C mRNA in U937 cells. When U937 (CD32+) cells are incubated with either PMA or IFN-gamma a significant enhancement of CD32C mRNA expression is observed with maximum enhancement at 18 hrs post-PMA and IFN-gamma addition. The addition of actinomycin D (ActD), a transcriptional inhibitor, together with either PMA or IFN-gamma diminishes the enhanced levels of CD32C mRNA to the basal levels, indicating that transcriptional regulation is involved in this modulatory process. The addition of cyclohexamide (CX), a protein synthesis inhibitor, to cultures undergoing stimulation with either PMA or IFN-gamma, increased the levels of CD32C mRNA synthesis suggesting that regulatory degradation proteins may be involved. The PMA and IFN-gamma stimulated CD32C mRNA is degraded within 2 hr post-stimulation and this degradation is delayed by the inhibition of de novo protein synthesis. These results, taken together with our previous studies of CD32A mRNA regulation in U937 cells stimulated with PMA, indicate that both the CD32A and C isomer mRNAs are rapidly degraded; however, CD32A and C isomer mRNAs are differentially regulated. At the optimal PMA dose, the time of mRNA stimulation of CD32A and C mRNA varies and the addition of CX to U937 cells together with PMA enhanced the levels of CD32C mRNA but had no effect on CD32A mRNA levels. These results imply that the differential regulation of the two CD32 isomers may result in differential function.

CD32A (Fc gamma RIIa) mRNA expression and regulation in blood monocytes and cell lines.

The cell surface expression of the CD32 receptors for the Fc portion of immunoglobulin G (Fc gamma RII) 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.

Structure of the gene for human von Willebrand factor.

von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes. Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene. A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries. The gene is approximately 178 kilobases in length and contains 52 exons. The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length. The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene. A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic. Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.

Regulation of human IgE synthesis by soluble factors. Papain treatment of a FcE receptor-positive B-cell line (RPMI-1788) releases regulatory factors for IgE synthesis.

A novel mechanism for the release of helper and suppressor factors for human IgE synthesis is described. When FcE receptor-positive RPMI-1788 cells are treated with papain, a helper factor(s) for human IgE synthesis is released. At the same time a significant decrease in the number of cell surface FcE receptors is observed. The immunoglobulin synthesis-enhancing activity is IgE isotype-specific inasmuch as the same supernatant suppresses the synthesis of human IgA myeloma cells. When the FcE receptor-positive RPMI-1788 cells are treated with tunicamycin and then with papain, a suppressor factor(s) for human IgE synthesis is released. The mechanism by which these factors affect human myeloma IgE synthesis is unclear at present. Our results indicate that enhanced IgE synthesis is not due to increased numbers of secreting cells nor to an increased release of presynthesized IgE. In summary, papain treatment of FcE receptor-positive, but not FcE receptor-negative cells, generates a factor that regulates IgE synthesis. These results also provide evidence for the close relationship between the IgE regulatory factors and the low affinity receptors for IgE present on lymphocytes.

Induction of human immunoglobulin synthesis (IgG, IgA) by the novel, T cell independent mitogen Cytophaga allerginae endotoxin.

Cytophaga allerginae endotoxin (CAE) has been purified from C. allerginae, a newly discovered bacterial species isolated from a chilled water spray humidification system. The present study was undertaken in order to determine whether CAE can induce immunoglobulin synthesis by human peripheral blood lymphocytes (PBL) in culture. To this end, human PBL were purified and cultured with either pokeweed mitogen at 5 micrograms/ml, or CAE (at varying concentrations) for 6 days. The levels of IgG and IgA in the supernatants were determined by the particle concentration fluorescence immunoassay and the IgE levels were determined by the avidin-biotin enzyme-linked immunosorbent assay. Our results indicate that CAE added to cultures in vitro induces IgG and IgA synthesis. CAE is a T cell independent mitogen inasmuch as ciclosporin A does not inhibit CAE-stimulated immunoglobulin synthesis but does inhibit PWM-stimulated immunoglobulin synthesis. In addition, CAE causes human B cell differentiation through the B1 and the DR determinants. In summary, CAE is a novel mitogen which can be used to induce human IgG and IgA synthesis in a T cell independent manner.

An avidin-biotin ELISA assay for the measurement of de novo human IgE synthesis in culture supernatants.

A very sensitive (100 pg/ml) solid-phase enzyme immunoassay (ELISA) for the determination of human IgE has been developed. This assay incorporates the avidin-biotin system to increase sensitivity and can detect as little as 100 pg/ml (10 pg/test) of human IgE. The assay is highly specific and allows quantitative determination of human IgE in supernatants of peripheral blood lymphocytes as well as in serum. The very high sensitivity of the assay was accomplished by optimizing concentrations of the following reagents: (1) affinity-purified rabbit anti-human IgE coating antibodies; (2) biotin-conjugated goat anti-human IgE; (3) avidin-horseradish peroxidase (HRP) conjugate. In summary, the assay described is rapid (6 h), reproducible, isotype specific, and has the sensitivity of radioimmunoassays usually employed for the quantification of IgE. This assay may be utilized in establishing concentrations of in vitro IgE levels synthesized by human peripheral blood lymphocytes (PBL).

Suppression of mixed lymphocyte culture by uremic adherent spleen cells and peritoneal macrophages is dependent on a cyclophosphamide-sensitive cell.

A model of experimentally induced uremia in the rat has been used to study the effect of uremia on the response of spleen cells to alloantigens. The proliferative ability of uremic spleen cells in mixed lymphocyte culture is significantly suppressed when compared to that of cells from control animals. This suppression appears to be due to both adherent suppressor cells which can be eliminated by adherence to rayon wool and to the inability of uremic T cells to respond to alloantigens. In addition, unstimulated peritoneal macrophages ( PMO ) obtained from uremic rats were also shown to be suppressive to the response of control spleen cells to alloantigens. The suppression by uremic adherent spleen cells and PMO is regulated by cyclophosphamide-sensitive cells.

The role of monocytes and responder cells in suppression of antigen-specific T cell proliferation in chronic renal failure.

The response of peripheral blood lymphocytes (PBL) to the antigen tetanus toxoid (TT) in patients with chronic renal failure being maintained on hemodialysis was examined. We have found that: (1) the response of patients' unfractionated PBL to TT is markedly suppressed when compared to the response of control PBL; (2) the response of patients' purified T cells and T4+ cells to TT co-cultured with 5% autologous monocytes is suppressed when compared to the response of comparable control cultures; (3) the response of patients' purified T lymphocytes co-cultured with 5% autologous monocytes is significantly enhanced over the response of patients' unfractionated PBL, and (4) the suppressed proliferation of patients' PBL to TT is not reversed by hemodialysis. Thus, the presence of suppressor monocytes and the inability of the responding T cells and accessory monocytes to react to antigen contribute to the suppressed antigen-specific T cell proliferation observed in chronic renal failure patients. These results are relevant to the suppressed cell-mediated immunity observed in uremia.

Immune response in experimentally induced uremia. VII. Uremic thymocytes amplify the response of control lymph node cells to alloantigens.

The effect of experimentally induced uremia in the rat on the synergistic response between thymus cells (TC) and lymph node cells (LNC) was examined. It was found that: (1) Uremic TC and LNC interact in a synergistic fashion which is greater than that observed for control cells; (2) The response of uremic LNC to alloantigens is suppressed when compared to the response of control LNC; (3) Uremic TC provide more help to control LNC in their response to alloantigens than do control TC; and (4) Treatment of uremic rats with cortisone acetate (CA) enhances their TC ability to amplify control LNC response to alloantigens. Thus, it appears that, while the response of uremic LNC to alloantigens is markedly suppressed, there are potent amplifier cells present in the thymus of uremic rats which have the ability to act in synergism with control LNC in response to alloantigens. This effect is significantly greater than the synergistic activity of control thymocytes.

Immune response in experimentally induced uremia. VI. Uremic macrophages are defective in their ability to present antigen to T cells.

The ability of uremic lymphocytes to respond to antigens was examined. We have found that (1) The ability of uremic unfractionated lymph node cells to respond to antigens is severely diminished when compared to the response of control cells; (2) Uremic macrophages are defective in their ability to present antigen to T cells; (3) Treatment of control splenic macrophages with monoclonal anti-Ia antibodies diminishes these macrophages' ability to present antigen, while uremic macrophages so treated show little change in their already diminished accessory cell function; and (4) The uremic splenic macrophage population has more small cells and less Ia determinants per cell than do control splenic macrophages as determined by cytofluorographic analysis. The percentage of Ia+ splenic macrophages is similar in control and uremic rats. It appears that the diminished response of uremic cells to antigens is due to the inability of uremic macrophages to present antigen to T cells. This may play a role in the increased rate of infections seen in uremic patients.

Immune response in experimentally induced uremia. V. Description of a model for chronic uremia in the rat that produces a long-term suppressive effect on T cell responses to mitogens.

This study describes a model for chronic uremia in the rat that produces a long-term suppressive effect on T cell responses to mitogens. We have found that (1) chronically uremic rats have significantly higher serum creatine levels than control rats and lymphocytopenia at all intervals tested after induction of uremia up to 4 1/2 months; (2) at all times tested after induction of uremia, the response of spleen cells from uremic rats to the T cell mitogens ConA and PHA was significantly suppressed as compared to control rats; (3) the severely suppressed response to PHA at all intervals tested after induction of uremia was eliminated by the removal of adherent spleen cells; and (4) adherent spleen cells, PMø, and AMø from chronically uremic rats are significantly more suppressive to control T cells than the corresponding control cells at all intervals tested after induction of uremia. Thus this animal model for chronic uremia is stable for at least 4 1/2 months and the effect of uremia on the response of lymphocytes to mitogens as well as on regulatory cells is not transient. Therefore this animal model can be used to study the effect of chronic uremia on the various parameters of the immune response.

Suppressor alveolar macrophages in experimentally induced uremia.

The effect of uremic alveolar macrophages (AM phi) on the response of control nonadherent (NA) spleen cells to mitogens was examined. Alveolar macrophages purified from experimentally induced uremic rats were found to be significantly more suppressive than alveolar macrophages purified from control rats. When studying the characteristics of uremic AM phi, it was found that neither indomethacin nor anti-Ia antibody reverses their suppressive activity. In addition, uremic AM phi are shown to suppress via a suppressor factor released to the supernatant over an 18-hr incubation. The enhanced suppressive activity of uremic AM phi may have relevance to the severely suppressed cell-mediated immunity, the increased rate of infections, and the common presence of uremic pneumonitis in humans with chronic renal failure.

Immune response in experimentally induced uremia. IV. Characterization of suppressor peritoneal macrophages in the uremic rat.

The effect of unstimulated uremia PM phi on the response of syngeneic control NA spleen cells to mitogens was examined. Nonstimulated PM phi purified from uremic rats were found to be significantly more suppressive than similar numbers of control PM phi. It was found that indomethacin treatment, as well as anti-la treatment, only partially reverses the suppressive activity of uremic PM phi as compared to control PM phi, thus indicating that uremic suppressor PM phi have different characteristics from control PM phi. In addition, uremic PM phi suppress via a suppressor factor released to the supernatant over 24 hr incubation. The enhanced suppressor activity of uremic PN phi as well as their different characteristics may have relevance to the severely suppressed cell-mediated immunity observed in uremia.