[Show abstract][Hide abstract] ABSTRACT: New functions are implicated for calreticulin, based on its release from cytotoxic NK and T cells. Calreticulin is the only
one of six “KDEL” (Lys-Asp-Glu-Leu carboxy terminal) endoplasmic reticulum chaperone proteins present in cytotoxic granules
of these lymphocytes. Calreticulin is released when the lymphocytes release their granules to kill other cells, which suggests
that it is likely to have an important role. In this chapter, we briefly review the contents of cytotoxic granules and how
calreticulin might interact with these proteins. Then we discuss calreticulin inactivation of lysis mediated by perforin,
a pore-forming protein which is essential for granule-mediated toxicity. Calreticulin is degraded by granzymes (proteases
found within the cytotoxic granules), particularly by one granzyme, Chymase 1. Chymase 1 was previously identified as required
for granule-mediated lysis, which could indicate that the calreticulin function(s) may be temporally regulated. We also introduce
our “inactivation of the inactivator” hypothesis (granzyme-degradation of the inactivator calreticulin) as a focus for evaluation
of the present information and as a focus to identify critical information to collect in the future. Finally, we provide the
reader with additional functions of calreticulin that could be important for systemic immunity.
[Show abstract][Hide abstract] ABSTRACT: IL-4 induces a lipase, pancreatic lipase related protein 2 (PLRP2), in cytotoxic T lymphocytes (CTLs). Because PLRP2 in semen can mediate lipid-dependent toxicity to sperm, we questioned whether CTL-derived PLRP2 could support similar cytotoxicity toward tumor cells. Recombinant PLRP2 was toxic to P815 tumor cells in 48 h when lipid and another protein, colipase, were present. However, PLRP2-positive CTLs (induced with many lots of IL-4) were unable to mediate lipid-dependent cytotoxicity. Notably, CTLs induced with only one lot of IL-4 had lipid-dependent cytotoxicity. The exceptional lot of IL-4 was effective in multiple experiments at inducing lipid-dependent cytotoxicity. The lipid-dependent cytotoxicity it induced was determined to be perforin-independent. CTLs induced with IL-4 that was unable to induce lipid-dependent cytotoxicity had mRNA for PLRP2 but not mRNA for colipase. Therefore, we added exogenous colipase to the CTL assays but still cytotoxicity was unchanged. We conclude (1) that lipid-dependent cytotoxicity, promoted by the lipase PLRP2 and colipase, will kill tumor cells and (2) that more than PLRP2 alone is required for lipid-dependent cytotoxicity mediated by CTLs.
Full-text · Article · Jul 2009 · Cell Biochemistry and Function
[Show abstract][Hide abstract] ABSTRACT: Pancreatic lipase-related protein 2 (PLRP2) is induced by IL-4 in vitro in cytotoxic T lymphocyte (CTL) clones and CTLs from immunized wild-type (WT) PLRP2(+/+) are more cytotoxic than PLRP2(-/-) CTLs, suggesting to previous investigators that the lipase PLRP2 might support CTL functions. Here, we further evaluate PLRP2 in CTLs. We found that PLRP2 was optimally induced in splenocytes by 3.5 x 10(-8) M IL-4 by day 6 after activation and was restricted to CD8(+) T cells. PLRP2 mRNA was detected inconsistently (and at low levels) after activation in the presence of IL-2. Cytotoxicity in 4 h (51)Cr assays of WT CTLs was approximately 3-fold the activity of PLRP2(-/-) CTLs cultured with IL-4 and, with IL-2, was unexpectedly approximately 2 fold the activity of PLRP2(-/-) CTLs. Thus, PLRP2 gene ablation affected short-term (perforin-dependent) cytotoxicity, even under the IL-2 conditions. Other variables failed to account for the reduced cytotoxicity. Granzyme B levels, activation markers, and CD8(+) T cell frequencies were similar for WT vs. PLRP2(-/-) CTLs (with either cytokine). Addition of rPLRP2 to IL-4 induced PLRP2(-/-) CTLs (or to cytotoxic granule extracts) failed to increase lysis, suggesting that the missing mediator is more than released PLRP2. Cytotoxicity of WT and PLRP2(-/-) CTLs was similar in 2-day tumor survival assays with IL-4, which can be mediated by perforin-independent mechanisms. We conclude that extracellular PLRP2 lipase is unable to directly augment the cytotoxicity that was lost by PLRP2 ablation and that after reevaluation, the question of what is PLRP2's role in CD8 T cells is still unanswered.
Full-text · Article · Jun 2009 · Journal of leukocyte biology
[Show abstract][Hide abstract] ABSTRACT: Contributions of lipases to CTL function have been debated, including if T cell lipases damage target cells. Expression of the lipase pancreatic lipase-related protein 2 (PLRP2) was previously found in IL-4 cultured lymphocyte cell lines but absent from IL-2 cultured lymphocytes. Here, we evaluated IL-2 and IL-4 induced CTLs for hydrolysis of target cell lipids and killing. Using anti-CD3 redirected lysis of [(3)H]-oleic acid-labeled P815 tumor cells, we detected the release of the radioactive fatty acid (FA). When PLRP2(+/+) and PLRP2(-/-) CTLs were compared, there was more killing by the PLRP2(+/+) CTLs. However, [(3)H]-oleic acid release was similar per dead P815, suggesting that lipid hydrolysis was produced by the dead P815s rather than by PLRP2. The FA release and death were completely dependent on perforin and also occurred when P815s were killed by perforin-containing T cell granule extracts that lacked lipase activity. Death by the cytotoxic granules extracts was unaffected by the addition of lipases. A lipase inhibitor, tetrahydrolipstatin, blocked FA release without affecting CTL-mediated cytotoxicity. Also, CTL-mediated death caused as much FA release as death by disruption of cells by freeze-thawing. The released oleic acid may be sufficient to promote secondary apoptotic responses after CTL-induced trauma.
Full-text · Article · Apr 2009 · International Immunology
[Show abstract][Hide abstract] ABSTRACT: Perforin, a membrane-permeabilizing protein, is important to T cell cytotoxic action. Perforin has potential to damage the T cell in the endoplasmic reticulum (ER), is sequestered in granules, and later is exocytosed to kill cells. In the ER and after exocytosis, calcium and pH favor perforin activity. We found a novel perforin inhibitor associated with cytotoxic T cell granules and termed it Cytotoxic Regulatory Protein 2 (CxRP2). CxRP2 blocked lysis by granule extracts, recombinant perforin and T cells. Its effects lasted for hours. CxRP2 was calcium stable and refractory to inhibitors of granzyme and cathepsin proteases. Through mass spectrometric analysis of active 50-100 kDa proteins, we identified CxRP2 candidates. Protein disulfide isomerase A3 was the strongest candidate but was unavailable for testing; however, protein disulfide isomerase A1 had CxRP2 activity. Our results indicate that protein disulfide isomerases, in the ER or elsewhere, may protect T cells from their own perforin.
Full-text · Article · Feb 2009 · Cellular Immunology
[Show abstract][Hide abstract] ABSTRACT: It is widely accepted that naïve T cells require two signals, antigen recognition and co-simulation, to become cytotoxic over the course of 3-5days. However, we observed that freshly isolated murine splenocytes without exposure to antigen become cytotoxic within 24h after culture with IL-15. IL-15 is a cytokine that promotes homeostatic proliferation, maintenance and activation of memory T cells. The induced cytotoxicity, measured by anti-CD3 redirected (51)Cr release, represented the combined activity of T cells regardless of their antigen specificity, and proceeded even when CD44(hi) (memory-associated phenotype) CD8(+) T cells were depleted. Cytotoxic capacity was perforin-dependent and occurred without detectable up-regulation of granzyme B or cell division. After induction, the phenotypic markers for the memory subset and for activation remained unchanged from the expression of resting T cells. Our work suggests that T cells may gain cytotoxic potential earlier than currently thought and even without TCR stimulation.
Full-text · Article · Mar 2008 · Cellular Immunology
[Show abstract][Hide abstract] ABSTRACT: Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.
Full-text · Article · Feb 2008 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: The purpose of these studies was to determine the minimal requirements to induce granzyme B, cytotoxic granules and perforin-dependent lytic capacity. To our surprise, both IL-2 and IL-15 induced not only proliferation, but also profound granzyme B and lytic capacity from CD8+ T cells in the absence of antigen or TCR-stimulation. Mouse splenocytes were incubated with mouse r-IL-2 or r-IL-15 for three days, tested by anti-CD3 redirected lysis and examined for intracellular granzyme B and for T cell activation markers. With 10(-8) M IL-2 or IL-15, there was excellent lytic activity at 1:1 effector to target ratios mediated by T cells from wild-type but not from perforin-gene-ablated mice, consistent with multiclonal activation. Lower interleukin concentrations induced less lytic activity. Granzyme B was undetectable on day 0, and greatly elevated on day 3 in CD44hi CD8+ T cells as detected by flow cytometry. Cytokines alone elevated the granzyme B as much as concanavalin A combined with the cytokines. Some ex vivo CD8+ T cells were CD122+, as were the cultured granzyme B+ cells, thus both populations had low-affinity receptors for the interleukins. Only some of the activated cells were proliferating as detected by CFSE labeling. When the cytokines were withdrawn, the cells lost lytic activity within 24 h and then within the next 24 h, died. Our results suggest that high concentrations of either IL-2 or IL-15 will activate the lytic capacity and granzyme B expression of many T cells and that antigen recognition is not required.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to examine the 100-fold difference in mycoplasma levels in lungs of gamma interferon knockout (IFN-gamma(-/-)) mice compared to those seen with wild-type BALB/c mice at 3 days postinfection. NK cells secreted IFN-gamma; however, their cytotoxic granule extracts failed to kill mycoplasma. We found a conundrum: the clearance of organisms was as effective in NK-depleted IFN-gamma(-/-) animals as in wild-type mice (with both IFN-gamma and NK cells). NK(+) IFN-gamma(-/-) animals had high mycoplasma burdens, but, after NK-like cell depletion, mycoplasma numbers were controlled. Essentially, IFN-gamma was important in animals with NK-like cells and unimportant in animals without NK cells, suggesting that IFN-gamma counters deleterious effects of NK-like cells. Impairment of innate immunity in IFN-gamma(-/-) mice was not due to NK-like cell killing of macrophages. The increased levels of inflammatory cytokines and neutrophils in lung fluids of NK(+) IFN-gamma(-/-) mice were reduced after NK cell depletion. In summary, in the murine model that resembles chronic human disease, innate immunity to mycoplasma requires IFN-gamma when there are NK-like cells and the positive effects of IFN-gamma counteract negative effects of NK-like cells. When imbalanced, NK-like cells promote disease. Thus, it was not the lack of IFN-gamma but the presence of a previously unrecognized NK-like cell-suppressive activity that contributed to the higher mycoplasma numbers. It appears that pulmonary NK cells may contribute to the immunosuppressive environment of the lung, but when needed, these dampening effects can be counterbalanced by IFN-gamma. Furthermore, there may be instances where perturbation of this regulatory balance contributes to the susceptibility to and severity of disease.
Full-text · Article · Nov 2005 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: Endothelial cell death induced by cytotoxic T cells is a key initiating event in the development of transplant vascular disease (TVD), the leading cause of late solid organ transplant failure. We studied the role of the granzyme B (GrB) pathwaye, which is one of the main mechanisms by which T cells induce apoptosis of allogeneic targets, in the pathogenesis of TVD. Granzyme B, in combination with perforin (pfn), induced apoptosis of cultured endothelial cells. In hearts transplanted into GrB knockout (GrB-KO) mice, there was a similar level of vasculitis as compared to WT mice, indicating that GrB does not affect immune infiltration into allograft arteries. However, there was a significant reduction in luminal narrowing of allograft arteries from GrB-KO mice as compared to WT recipients. These results indicate that GrB plays a role in endothelial cell death in allograft arteries and in the resultant development of TVD.
Full-text · Article · Apr 2005 · American Journal of Transplantation
[Show abstract][Hide abstract] ABSTRACT: Dipeptidyl peptidase I (DPPI, cathepsin C) is a lysosomal cysteine protease that can activate zymogens of several different serine proteases by one step or sequential removal of dipeptides from the N-termini of the pro-protease protein substrates. To find DPPI inhibitors more suitable for cellular applications than diazomethyl ketones, we synthesized three types of inhibitors: dipeptide acyloxymethyl ketones, fluoromethyl ketones, and vinyl sulfones (VS). The acyloxymethyl ketones inhibited DPPI slowly and are moderate inhibitors of cellular DPPI. The fluoromethyl ketones were potent, but the inhibited DPPI regained activity quickly. The dipeptide vinyl sulfones were effective inhibitors for DPPI, but they also inhibited cathepsins B, H, and L weakly. The best inhibitor, Ala-Hph-VS-Ph, had a k2/K(I) of 2,000,000M(-1)s(-1). The vinyl sulfones also inhibited intracellular DPPI, and for this application the more stable inhibitors exhibit better potency. We conclude that vinyl sulfones are promising inhibitors to study the intracellular functions of DPPI.
Full-text · Article · Aug 2004 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: Optimal differentiation of cytotoxic NK cells is important to provide protective innate immunity to patients after bone marrow transplantation. In vitro differentiation of CD56(+)CD3(-) NK cells takes weeks and is supported by several cytokines, including IL-2, IL-7, and IL-15, and thus can be useful for immunotherapy. However, IL-2 therapy is problematic in vivo, and NK cells differentiated in vitro with only IL-7 lack cytotoxicity. We assessed whether human NK cells initially differentiated in vitro from CD34(+)Lin(-) bone marrow cells with IL-7 could acquire cytotoxicity after exposure to additional cytokines and what changes promoted cytotoxicity. The cells cultured with IL-7 already had granzyme B as well as perforin, as previously reported, the proteins of cytotoxic granules. The cells also lacked LFA-1. After 1 wk of secondary culture with either IL-2 or IL-15, but not with IL-12 or IL-18, the IL-7-cultured cells acquired cytotoxicity. IL-2 or IL-15 also induced LFA-1. Ab to the LFA-1 subunits CD11a and CD18 blocked lysis by the NK cells, indicating that the new LFA-1 correlated with, and was essential for, the cytotoxic function of the in vitro generated cells. The LFA-1 also participated in target cell binding by the in vitro differentiated cells. In this study, we demonstrated a new function for IL-15, the induction of LFA-1 in NK progenitor cells, and that IL-15 does more than merely support NK progenitor cell proliferation. The efficacy after only 1 wk of IL-15 administration is a positive practical feature that may apply to human therapy.
Full-text · Article · Aug 2003 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: The broadly reactive cysteine protease dipeptidyl peptidase I (DPPI, cathepsin C) is thought to activate all progranzymes (zymogens of lymphocyte serine proteases) to form mature granzymes. We synthesized dipeptide 7-amino-4-methylcoumarin (AMC) substrates containing progranzyme activation sequences and showed that they were efficiently hydrolyzed by DPPI. However, DPPI will not hydrolyze Ile-Ile-AMC, the N-terminal dipeptide sequence found in mature granzymes. Introduction of the nonphysiological homophenylalanine (Hph) residue at P1 resulted in the best substrate Ala-Hph-AMC for DPPI (k(cat)/K(m)=9,000,000M(-1)s(-1)). The charged N-terminal amino group of the substrate was essential and replacement of the NH(2) group with OH or NH(CH(3)) in Gly-Phe-AMC reduced the k(cat)/K(m) value by two to three orders of magnitude. A hydrazide azaglycine analog, NH(2)NHCO-Phe-AMC, was not hydrolyzed at pH 5.5, but underwent slow hydrolysis at lower pHs where the amino group is partially protonated. DPPI also failed to hydrolyze NH(2)COCH(2)-Phe-AMC, where the NH(2) group is unprotonated. The results reported in this paper should be useful in the design of better DPPI inhibitors to block granzyme maturation and granzyme-dependent apoptosis.
No preview · Article · Aug 2002 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: Quantitative immunophenotypes varied widely among > 100 healthy young males but were maintained at characteristic levels within individuals. The initial results (SPIE Proceedings 4260:226) that examined cell numbers and the quantitative expression of adhesion and lineage-specific molecules, e.g., CD2 and CD14, have now been confirmed and extended to include the quantitative expression of inducible molecules such as HLA-DR and perforin (Pf). Some properties, such as the ratio of T helper (Th) to T cytotoxic/suppressor (Tc/s) cells, are known to be genetically determined. Other properties, e.g., the T:B cell ratio, the amount of CD19 per B cell, etc., behaved similarly and may also be inherited traits. Since some patterns observed in these healthy individuals resembled those found in pathological situations we tested whether the patterns could be associated with the occurrence of disease. The current studies shows that there were associations between quantitative immunophenotypes and the subsequent incidence and severity of disease. For example, individuals with characteristically low levels of HLA-DR or B cells or reduced numbers of Pf+ Tc/s cells had more frequent and/or more severe upper respiratory infections. Quantitative immunophenotypes will be more widely measured if the necessary standards are available and if appropriate procedures are made more accessible.
No preview · Article · May 2002 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: In granules of hematopoetic cells, dipeptidyl peptidase I (DPPI) processes inactive proenzymes into active enzymes, e.g., lymphocyte progranzyme A. Our goal was to develop irreversible inhibitors of intracellular DPPI. First, we identified inhibitors with aqueous stability. Then we determined which inhibitors were nontoxic, could enter cells and inactivate intracellular DPPI. We screened nine dipeptide vinyl sulfone (VS) inhibitors (kobs/[I] > 72 M-1 s-1) and found six that were nontoxic. Four affected intracellular DPPI at < 25 microM. These compounds contained only uncharged amino acid residues; the two less reactive compounds contained charged Glu residues. The best one, Leu-Phe-VS-CH3, inactivated DPPI in cells with an ID50 of approximately 5 microM. This inhibitor was not the best inhibitor of purified DPPI. Longer aqueous stabilities were important predictors of cellular efficacy. Leu-Phe-VS-CH3 had a half life of 97 min at the pH of the extracellular medium (7.5) and 1302 min at pH 5.5 (the intracellular environment of DPPI). This VS had no direct effect on granzyme activities. In contrast, the diazomethyl ketone inhibitor Gly-Phe-CHN2 inhibited chymase activity. Several good intracellular DPPI VS inhibitors lacked reactivity with cathepsins B, H and L. In conclusion, we have identified DPPI inhibitors suitable for cellular applications.
No preview · Article · Jan 2001 · International Immunopharmacology
[Show abstract][Hide abstract] ABSTRACT: The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 x 10-7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8. 8 x 10-7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.
Full-text · Article · May 2000 · The Journal of Immunology