Preventive usage of broad spectrum chemokine inhibitor NR58-3.14.3 reduces the severity of pulmonary and hepatic graft-versus-host disease
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- Miklos, S., Mueller, G., Chang, Y. et al. Int J Hematol (2009) 89: 383. doi:10.1007/s12185-009-0272-y
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Pulmonary graft-versus-host disease (pGVHD) is a major complication after allogeneic bone marrow transplantation (BMT), which involves donor leukocyte migration into the lung along chemokine gradients, leading to pulmonary dysfunction and respiratory insufficiency. As broad spectrum chemokine inhibitor (BSCI) NR58-3.14.3 suppresses leukocyte migration in response to various chemokines, including CCL2, CCL3, CCL5, we investigated the effects of NR58-3.14.3 on the evolution of pGVHD. Lethally irradiated B6D2F1 mice received BMT from syngeneic (B6D2F1) or allogeneic (C57BL/6) donors, and animals were treated with either NR58-3.14.3 or vehicle control from day −1 to day +14. At week 6, in allogeneic recipients that received BSCI, inflammatory cell infiltrates in the lung were decreased, and reduced histopathologic changes translated into improved pulmonary function when compared to allo-controls. Acute GVHD of the liver was also diminished, whereas no differences were seen in the gut. Alloantigen-dependent splenic T cell expansion and systemic TNF-α and IFN-γ levels were comparable in NR58-3.14.3-treated animals and allo-controls. No suppressive effect of NR58-3.14.3 on CTL cytotoxicity was found, and diminished cellular infiltrates in lung and liver were most likely due to decreased migration of mononuclear cells. Therefore, novel approaches involving BSCIs may provide a promising tool in the management of pGVHD.
KeywordsLungAllogeneic bone marrow transplantationChemokineGraft-versus-host diseaseIdiopathic pneumonia syndrome
Allogeneic bone marrow transplantation
Acute graft-versus-host disease
Antigen presenting cell
Broad spectrum chemokine inhibitor
Cytotoxic T lymphocyte
Forced expiratory flow
Forced expiratory volume
Intercellular adhesion molecule 1
Idiopathic pneumonia syndrome
Major histocompatibility complex
Pulmonary function testing
Pulmonary graft-versus-host disease
Standard error of the mean
Total body irradiation
Tumor necrosis factor alpha
Allogeneic bone marrow transplantation (allo-BMT) is an important therapy for a number of malignant and non-malignant diseases . However, its utility is limited by severe and potentially lethal complications including the development of acute graft-versus-host disease (aGVHD) and pulmonary toxicity [2–7]. Acute non-infectious diffuse lung injury, classically defined as idiopathic pneumonia syndrome (IPS), has been associated with mortality rates of >70% [2, 5] and an incidence between 5 and 25%, depending on potential risk factors such as conditioning regimen intensity, donor source and antigenic mismatch between donor and recipient [2–7]. The pathophysiology of both IPS and aGVHD involves toxic damage due to irradiation and chemotherapy [8, 9], an allospecific donor T cell response [10–13], and the production of inflammatory cytokines [11, 14–18]. Although non-infectious pulmonary injury after allo-BMT has not been traditionally considered to be a form of acute or chronic GVHD, data from murine and human studies showed that the lung is a critical target organ of alloimmune responses [10, 19–21], leading to the concept of IPS as a form of pulmonary graft-versus-host disease (pGVHD) .
Infiltration of the lung by donor T cells, monocytes and macrophages is a hallmark of alloimmune-mediated pulmonary injury [10–14, 22]. The recruitment of cellular effectors to sites of inflammation is strongly dependent upon the establishment of chemokine gradients in the inflamed tissue. Therefore, chemokine expression profiling and mechanistic studies looking at the interactions between specific chemokines and their respective receptors in the context of IPS development have been a strong focus of attention [22–29]. Initially, it was shown that several pro-inflammatory chemokines are increasingly expressed in the lung early after allo-BMT , followed by subsequent demonstration of a functional relevance of specific chemokine ligand–chemokine receptor interactions for full development of IPS [22, 23, 25, 26, 29]. In these studies, the severity of IPS was decreased by individually targeting CXCR3, CCR2, CCL2 (MCP-1), CXCL9 (Mig), CXCL10 (IP-10) or CCL5 (RANTES) [22–25].
Grainger et al.  have recently developed a series of oligopeptides that act as functional chemokine inhibitors. One of these oligopeptides is the broad spectrum chemokine inhibitor (BSCI) NR58-3.14.3, a powerful anti-inflammatory agent, which suppresses the in vitro and in vivo migration of leukocytes in response to several chemokines including CCL2, CXCL8 (IL-8), CCL3 (MIP-1α) and CCL5 .
In light of the established role of alloreactive lymphocytes and donor accessory cells including monocytes and macrophages in the development of IPS [10–12, 14, 23, 25], we hypothesized that blocking a broader range of chemokines using a BSCI rather than targeting only one specific chemokine or chemokine receptor would comparably or further reduce the development of pGVHD after allo-BMT. We used a well-established murine BMT model, wherein the allospecific graft-versus-host response is mediated by donor CD4+ and CD8+ T lymphocytes to MHC class I, MHC class II and minor Hag mismatches between donor and host and which results in the reliable induction of lung injury. Our data show that treating allogeneic recipients with NR58-3.14.3 during the first 2 weeks after transplantation results in improved pulmonary function and decreased pGVHD severity.
2 Materials and methods
2.1 Mice and bone marrow transplantation
Female C57BL/6 (H-2b) and B6D2F1 (H-2bxd) mice were purchased from Charles River Laboratories (Sulzbach, Germany) and acclimatized in our animal facility for at least 1 week before the experiments. Animals were between 10 and 20 weeks old at the time of BMT. All animal experiments were approved by the local institutional animal committee of the University of Regensburg and were in accordance with German animal protection laws.
Mice were transplanted according to a standard protocol as previously described . On the day of BMT, B6D2F1 recipient mice received lethal total body irradiation (TBI) delivered in two fractions 3 h apart to reduce gastrointestinal toxicity. TBI dose was 12 Gy; linear accelerator, 150 cGy/min. TBI was followed by the infusion of cell mixtures of 5 × 106 bone marrow cells supplemented with 6 × 106 splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. Broad spectrum chemokine inhibition was achieved in allogeneic recipients by subcutaneous injections of the BSCI NR58-3.14.3 (Funxional Therapeutics, Cambridge, UK) at a dose of 30 mg/kg body weight , dissolved in 100 μl saline, twice a day from day −1 to day +5 and once daily from day +6 until day +14. Single dose treatment of mice with NR58-3.14.3 at a dose of 30 mg/kg body weight once daily has been previously shown to be sufficient in vivo . As early chemokine expression after BMT promotes the development of pulmonary pathology [24, 27], we additionally decided to administer NR58-3.14.3 twice daily before and during the early phase after BMT to counterbalance increased induction of chemokine expression due to pro-inflammatory cytokine release. Allogeneic and syngeneic controls received equal amounts of saline following the same schedule of administration. In initial experiments, syngeneic recipients being treated with NR58-3.14.3 were also included to assess drug toxicity and side effects such as premature mortality due to infection. For determination of chemokine gene expression in GVHD target organs, animals received lethal irradiation (13 Gy) followed by syngeneic or allogeneic BMT without treatment with NR58-3.14.3. Transplanted mice were housed in micro-isolator cages with autoclaved bedding and received autoclaved chow and hyperchlorinated water ad libitum.
2.2 Clinical GVHD and survival
Survival was monitored daily until day +42 after BMT, and clinical GVHD scores were assessed weekly using a well-established scoring system incorporating five clinical parameters: weight loss, posture (hunching), mobility, fur texture and skin integrity as described previously . Each parameter was graded between 0 and 2. Once an animal reached a cumulative score ≥6.5 or a weight loss of more than 30%, it was killed and counted for analysis as transplantation-related mortality.
2.3 Pulmonary function analysis
Assessment of pulmonary function was performed using a Buxco lung function analysis system (BUXCO Electronics, Troy, NY) consisting of a pulmonary function test/forced maneuvers analyzer (SFT3840) plus pressure panel for mouse maneuvers (AUT6100), and an anesthetized mouse–PFT plethysmograph (PLY3112). Data acquisition and analysis were done using BioSystem XA software (SFT3850).
The anesthetized mouse–PFT plethysmograph allows the assessment of lung volumes and forced ventilation parameters. Measurements were performed according to the manufacturer’s on-site instruction and protocol. To perform these tests, animals were anesthetized by the intraperitoneal injection of ketamine hydrochloride (50 mg/kg) and medetomidine hydrochloride (0.33 mg/kg). The trachea was dissected, cleaned of surrounding tissue and cannulated with a 17-19 gauge cannula, depending on the trachea size and the size of the mouse. Animals were placed into the chamber and mouse lungs were connected over the tracheal cannula through a port in the chamber wall to a built-in ventilator. Sedation was ensured to be deep enough to prevent spontaneous breathing against passive ventilation before tests were performed. Fast flow volume measurements were done to assess air outflow obstruction and quasistatic pressure volume measurements were performed to assess lung volumes. Parameters included in the analysis were vital capacity (VC, μl), forced expiratory volume (FEV, μl) at 20 ms, forced expiratory flow (FEF, ml/s) at 25% of expiration and chord compliance at 0–10 cmH2O (Cchord, ml/cmH2O). Measurements were conducted 42 days after transplantation.
2.4 Semi-quantitative histopathology of lung, liver and intestine
On day +42 after transplantation, animals were killed for histopathological analysis. Organs were removed, fixed in 4% paraformaldehyde dissolved in PBS, transferred into 70% ethanol after 48 h, paraffin embedded and then sectioned into 4 μm slices. Hematoxylin–eosin-stained lung sections from individual mice were coded without reference to mouse type and prior treatment and independently examined to establish an index of injury. Lungs were evaluated for the presence of periluminal infiltrates or parenchymal pneumonitis using a semi-quantitative scoring system as described previously, including the severity (periluminal infiltrates: 0 = no infiltrates, 1 = 1–3 cell diameters thick, 2 = 4–10 cell diameters thick, 3 = >10 cell diameters thick; pneumonitis: 0 = no infiltrates, 1 = increased cells, only visible at high magnification, 2 = easily visible cellular infiltrate or interstitial thickening, 3 = consolidation by inflammatory cells and interstitial thickening) and extent (percentage of lung tissue involved: 1 = 5–25%, 2 = 25–50%, 3 = >50%) of histopathologic changes .
Detailed gastrointestinal tract and liver histopathologic analyses were also performed in a blinded fashion 42 days after transplantation as previously described [10, 34, 35]. Analysis was performed by light microscopy (Axioskop 2 plus, Carl Zeiss GmbH, Jena, Germany). Photographs of histopathology were acquired with the AxioCam HRc (Carl Zeiss GmbH) and processed with AxioVision Release 4.6.3 (Carl Zeiss GmbH).
2.5 Splenic T cell expansion and serum cytokine analysis after BMT
Spleens were harvested 7 days after transplantation and single cell suspensions were generated from individual animals. Splenocytes were subsequently counted and stained for CD4 and CD8. For cytokine analysis, animals were exsanguinated 7 days after BMT, and blood samples were collected in 1.5-ml Eppendorf tubes (Eppendorf, Hamburg, Germany) and centrifuged at 10,000 rpm for 10 min at 4°C. Serum supernatants were harvested for subsequent analysis for TNF-α and IFN-γ by ELISA as described below.
2.6 MLR cell culture and cytotoxic T lymphocyte (CTL) function
All cell culture reagents were purchased from Invitrogen Life Technologies (Karlsruhe, Germany) or from Sigma-Aldrich (Taufkirchen, Germany). Cell cultures were kept at 37°C in a humidified incubator supplemented with 5% CO2.
T cell proliferation in response to alloantigen was measured by co-culturing 2 × 105 splenic T cells from C57BL/6 mice in flat bottom, 96-well Falcon plates with 5 × 105 irradiated (30 Gy) B6D2F1 splenocytes in the presence of different concentrations of NR58-3.14.3 (10–100 μmol). T cells were isolated by magnetic bead separation using the OctoMACS system (Miltenyi, Bergisch Gladbach) according to the manufacturer’s protocol. Proliferative responses were measured using a Microplate Scintillation counter Topcount (Packard Canberra, Dreieich) after 48 h +16 h, 72 h + 16 h, 96 h + 16 h by incorporation of [3H]thymidine (1.9 × 105 Bq/ml) for the last 16 h of incubation. To measure proliferation of T cells in response to mitogen, splenic T cells were cultured in flat bottom, 96-well Falcon plates in the presence or absence of 3 μg/ml concanavalin A (Sigma, St Louis, MO, USA). The proliferative response was measured after 72 h by assessing incorporation of [3H]thymidine (1.9 × 105 Bq/ml) added for the last 16 h of incubation. Supernatants were harvested at the time of [3H]thymidine supplementation and analyzed for IFN-γ as described below.
Cytotoxic T lymphocyte assays were performed according to a previously published method using a 51Cr release assay . The P815 (H-2d) mouse mastocytoma cell line and the EL-4 cell line (H-2b) were used as allogeneic and syngeneic target cell lines, respectively. Target cells were labeled with 3.7 MBq of 51Cr at 37°C for 90 min and co-cultured with pre-activated effector T cells at different effector:target ratios (E:T ratio) in the presence or absence of NR58-3.14.3 (10 μmol). Chromium release was measured after 4 h of incubation and percentages of specific lysis for different effector:target ratios were calculated as (experimental release − spontaneous release)/(maximal release − spontaneous release) × 100%. Using a second approach, alloantigen-dependent priming of naïve T cells occurred in the presence or absence of NR58-3.14.3, whereas NR58-3.14.3 was not added during the chromium release assay itself.
2.7 Lung, liver and gut chemokine analysis
Following exsanguination organs were harvested 7 days after transplantation and immediately snap-frozen in liquid nitrogen. At the time of analysis, 70 mg of organ tissue was homogenized in 1 ml buffer solution (1× phosphate-buffered saline [PBS] and 1 tablet of Complete Protease Inhibitor Cocktail [Roche Diagnostics, Basel, Switzerland]), centrifuged at 1,500 rpm for 15 min at 4°C, and supernatant was harvested. Total protein concentration in the supernatant was determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) before chemokines were measured by ELISA as described below. Chemokine concentration in the supernatant was normalized to picograms per milligram of total protein.
2.8 Measurements of chemo- and cytokine levels by ELISA
Concentrations of chemo- and cytokines were measured in serum of transplanted animals, in cell culture supernatants or in supernatants from homogenized organs using ELISA (TNF-α and IFN-γ: OptEIA, BD Pharmingen, Heidelberg, Germany; CCL2, CCL3, CCL5, CXCL1 (KC) and CXCL2 (MIP-2): Quantikine M, R&D Systems GmbH, Wiesbaden, Germany) or cytokine bead array (BD Pharmingen). Assays were performed according to manufacturer’s protocols. ELISA plates were analyzed with a microplate reader (Emax, Molecular Devices, Sunnyvale, CA, USA).
2.9 Cell surface phenotype analysis and assessment of engraftment
Bone marrow was harvested by obtaining single cell bone marrow suspensions from flushed tibias and femurs on day 42 after BMT. Major histocompatibility complex (MHC) class I expression was analyzed to address engraftment. Bone marrow cells were stained with fluorescein isothiocyanate (FITC)-conjugated mAb to H-2kb and phycoerythrin (PE)-conjugated mAb to H-2kd for flow cytometric analysis. All mAbs were purchased from BD Pharmingen (San Diego, CA, USA). Two color flow cytometric analysis of 1 × 106 cells was performed using a FACSCalibur (BD Bioscience, San Jose, CA).
2.10 Determination of chemokine mRNA expression in organs
At defined time points after BMT, animals were killed, and following exsanguination organs were harvested. Total cellular RNA was extracted using RNeasy Mini Kits (Qiagen, Hilden, Germany). First-strand cDNA was synthesized using 1 μg of total RNA (DNase-treated) in a 20 μl reverse transcription. The reaction was performed as follows: 1 μg total RNA in 8 μl RNase free water was denatured for 5 min at 75°C, then 10 μl RT reaction mixture (Invitrogen, Karlsruhe, Germany) and 2 μl RT enzyme mixture (Invitrogen, Karlsruhe, Germany) were added. The RNA and reverse transcription mixture was incubated 10 min at 25°C followed by 60 min at 44°C and a denaturing step for 5 min at 85°C. The cDNAs were diluted 1/20 and stored at −20°C until use for real-time PCR.
All real-time PCR reactions were performed in a 10 μl mixture containing 4 μl of sample, 5 μl Sybr green mix (Invitrogen, Karlsruhe, Germany), 0.25 μl sense primer (10 μM), 0.25 μl antisense primer (10 μM) and 0.5 μl water. Real-time PCR quantifications were performed using the ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, Foster City, USA). The fluorescence threshold value was calculated using the ABI PRISM® 7900HT Sequence Detection System software version SDS 2.2. GAPDH was used as a reference. Primers used for real-time PCR were as follows: GAPDH sense, CAT CAC TGC CAC CCA GAA GA; GAPDH antisense, CAG ATC CAC GAC GGA CAC AT; CCL2 sense, GCT GAC CCC AAG AAG GAA TG; CCL2 antisense, GTG CTT GAG GTG GTT GTG GA; CCL3 sense, CCA CTG CCC TTG CTG TTC TT; CCL3 antisense, GGA GCA AAG GCT GCT GGT TT; CCL5 sense, CTC ACC ATA TGG CTC GGA CA; CCL5 antisense, CTT CTC TGG GTT GGC ACA CA; CXCL1 sense, GCC TAT CGC CAA TGA GCT G; CXCL1 antisense, CTG AAC CAA GGG AGC TTC AG G; CXCL2 sense, GGC TGT TGT GGC CAG TGA A; CXCL2 antisense, GCT TCA GGG TCA AGG CAA AC.
2.11 Statistical considerations
All values are expressed as mean ± standard error of the mean (SEM). Statistical comparisons between groups were completed using the parametric independent sample t test with five or more samples per group and the Mann–Whitney U test if n < 5 per group. p values were two sided and a level of p ≤ 0.05 was considered significant.
3.1 Determination of chemokine mRNA expression levels in GVHD target organs
Chemokine mRNA expression levels in GVHD target organs
3.95 ± 1
39.87 ± 5.72**
5.49 ± 0.94
47.85 ± 13.62**
0.57 ± 0.09
4.13 ± 0.49**
1.89 ± 0.6
15.75 ± 2.77**
0.32 ± 0.05
3.42 ± 0.58**
2.13 ± 0.64
14.93 ± 3.89**
1.46 ± 0.22
6.93 ± 0.99**
3.65 ± 0.5
7.81 ± 0.92**
0.99 ± 0.15
3.52 ± 0.65**
3.52 ± 0.55
7.25 ± 2.25
1.87 ± 0.28
28.64 ± 6.91**
1.42 ± 0.46
10.11 ± 2.65**
1.97 ± 0.61
29.34 ± 11.02**
0.83 ± 0.33
47.69 ± 11.42**
0.35 ± 0.07
5.54 ± 0.99**
0.7 ± 0.28
18.93 ± 4.66**
5.8 ± 0.77
233.16 ± 0.74**
3.16 ± 1.28
27.74 ± 6.17*
0.83 ± 0.14
8.43 ± 3.42**
2.05 ± 0.89
10.67 ± 4.33*
2.82 ± 0.57
17.52 ± 4.15**
4.26 ± 0.93
12.36 ± 2.37*
2.52 ± 0.63
145.32 ± 9.03**
2.5 ± 1.29
56.98 ± 26.16**
0.26 ± 0.09
7.94 ± 2.06**
1.69 ± 0.76
6.79 ± 1.53**
2.38 ± 0.72
8.79 ± 2.21*
2.87 ± 0.74
2.5 ± 0.49
18.68 ± 5.51
28.09 ± 5.9
7.03 ± 2.51
1.95 ± 0.53
3.2 Preventive treatment with NR58-3.14.3 decreases the severity of pGVHD after allo-BMT
3.3 Effects of NR58-3.14.3 on GVHD target organ injury in GI tract and liver
The severity of clinical GVHD was monitored over 6 weeks after transplantation. Due to radiation conditioning toxicity, syngeneic recipients demonstrated minor changes in weight loss, fur texture and mobility by week 1, but then rapidly regained body weight and completely recovered clinically. Allogeneic control-treated recipients showed severe weight loss and extensive symptoms of clinical GVHD after BMT. Allogeneic animals receiving NR58-3.14.3 also developed signs of moderate clinical GVHD, which, in extent and severity, were significantly reduced following week 2 (Fig. 2e, f), but did not translate into significant improvement of survival until week 6 when directly compared to allogeneic controls (57 vs. 52%) (Fig. 2g).
3.4 Effects of treatment with NR58-3.14.3 on alloreactive T cell responses
Reduced inflammatory cell infiltrates in lung and liver of allogeneic recipients treated with NR58-3.14.3 indicate that inhibition of leukocyte migration may be the major mechanism contributing to decreased pulmonary and hepatic injury. This is supported by previous reports, which showed strong inhibitory properties of NR58-3.14.3 on leukocyte migration in vitro and in vivo . In addition, various chemokines can promote alloreactive T cell responses [44, 45], and it could not be excluded that treatment with NR58-3.14.3 alters T cell activation and T cell function as well. We first tested the effects of NR58-3.14.3 on alloantigen-specific T cell proliferation and cytokine production by comparing the proliferative capacity of C57BL/6 T cells (H-2b) against allogeneic B6D2F1 (H-2bxd) antigen presenting cells or against ConA stimulation in the absence or presence of NR58-3.14.3 (10, 100 μM) as described in Sect. 2. Alloantigen-independent T cell stimulation with the lymphocyte mitogen ConA was included to test for effects of NR58-3.14.3 on nonspecific T cell proliferation. In addition, we controlled for intrinsic T cell activating properties of NR58-3.14.3 in the absence of alloantigens, using a syngeneic setting of B6D2F1 T cells (H-2bxd) against B6D2F1 (H-2bxd) stimulators.
As the reactivity of donor CTL’s against malignant cells is essential for graft-versus-leukemia (GVL) and graft-versus-tumor (GVT) effects, we next tested if treatment with NR58-3.14.3 altered CTL function of alloantigen-activated T cells in vitro. After co-culture of C57BL/6 T (H-2b) cells with B6D2F1 (H-2bxd) stimulators for 5 days, target cell lysis of either EL4 (H-2b) or P815 (H-2d) cells in the presence or absence of NR58-3.14.3 was assessed. NR58-3.14.3 had no effect on the lysis of allogeneic P815 targets (Fig. 3g), and neither was lysis induced when EL-4 targets were used as syngeneic controls (Fig. 3g). Similar results were obtained when T cell effectors were initially co-cultured with allogeneic stimulators in the presence of NR58-3.14.3 and no NR58-3.14.3 was added during the phase of target cell lysis (Fig. 3h).
3.5 Effects of NR58-3.14.3 on chemokine expression in GVHD target organs
3.6 NR58-3.14.3 does not impair engraftment after allo-BMT
NR58-3.14.3 does not impair engraftment after allo-BMT
Donor chimerism (%)
Mean ± SEM
96.2 ± 0.8
96.8 ± 0.3
Previous studies have shown a mechanistic role of specific chemokines and chemokine receptors for the recruitment of donor T cells, monocytes, and macrophages to the lung, contributing to the development of IPS [12, 22–26]. In most of these studies, lung injury was reduced, but not completely abrogated, which in part may present one of the limitations of blocking a single chemokine or chemokine receptor due to promiscuity within the chemokine network. Broad spectrum chemokine inhibition, therefore, seems an attractive therapeutic approach. Recently, the group of Dr. Grainger developed a series of oligopeptides that act as functional chemokine inhibitors. The chemokine inhibitor used in this study, NR58-3.14.3, is a D amino acid peptide that is restrained by intermolecular disulfide linkage, and is derived from a conserved region at the C terminus of CCL2. NR58-3.14.3 inhibits leukocyte migration in response to a range of CXC and CC chemokines, including CCL2, CCL5, CCL3 and CXCL8  and has been successfully used to treat various diseases in experimental animal studies [32, 41, 42, 48–51]. At present, the exact molecular mechanisms of NR58-3.14.3 responsible for the functional inhibition of chemokine-induced migration remain unknown. NR58-3.14.3 does not block chemokine binding to their receptors, nor does it bind to chemokine receptors itself . Recently, a cell surface receptor has been discovered as a molecular target for BSCI activity, and NR58-3.14.3, which acts agonistic at this site, generates a series of intracellular signals which effectively blind the cell to the directional component of chemokine signaling (Dr. Grainger, personal communication).
The association of pulmonary T cell infiltration with impaired pulmonary function after allo-BMT has been demonstrated in earlier studies [10, 12, 21]. In the present study, preventive treatment of allogeneic recipients with NR58-3.14.3 during the first 2 weeks after allo-BMT resulted in decreased pGVHD along with improved pulmonary function and decreased liver damage, but not in a reduction of gut GVHD. As shown by two independent groups, the tropism of T cell-mediated target organ injury to the lung after allo-BMT is partially regulated by the expression of adhesion molecules such as ICAM-1 [13, 52]. ICAM-1 deficient recipients developed significantly reduced lung injury after allo-BMT, associated with decreased T cell infiltration and improved pulmonary function [13, 52], whereas the severity of intestinal and hepatic GVHD was unchanged or increased [13, 52]. This indicates that mechanisms of leukocyte recruitment differ between GVHD target organs, and as chemokine–chemokine receptor interactions are major factors in directing cell migration and chemotaxis, the preferential recruitment of donor leukocytes to certain target organs may additionally relate to distinct chemokine expression patterns [53, 54]. In this study, strongest increases in CCL2 expression were seen in the lung, followed by liver and gut. Interestingly, a prior study showed that allo-BMT with donor cells, which lack the expression of CCR2 as the receptor for CCL2, resulted in significantly reduced lung pathology by week 6 compared with allogeneic CCR2+/+ controls, whereas aGVHD-associated target organ injury to liver or gastrointestinal tract and survival were comparable in the two groups . Donor T cells infiltrating the lung early after allo-BMT promote additional T cell and monocyte/macrophage recruitment through their secretion of CCL5 and drive the development of progressive lung disease without affecting GVHD of liver and gut . Similarly, donor T cell production of CCL3 also contributes to the recruitment of CD8+ T cells to the lung, but in addition it promotes CD8+ T cell infiltration of the liver [26, 55]. Consistent with increasing numbers of organ-infiltrating donor T cells , which secrete CCL3 and CCL5 as positive feedback mechanism [25, 26], in our study, hepatic and pulmonary expression of both chemokines rose over time. Gastrointestinal CCL3 seems predominantly expressed by host tissue cells, as it does not depend on the production by donor T cells , and, in our study, it was expressed highest during the first week after BMT, most likely mirroring its induction through inflammatory cytokines such as TNF-α . Receptors for CCL3 and CCL5 are CCR1 and CCR5. Comparable to the clinical setting, in conditioned mice, CCR5−/− T cell infiltrates in the GI tract, lung or liver were equal or even increased [23, 28, 56], whereas CCR1 deficiency of donor cells resulted in decreased cellular infiltration in all three organs [45, 57]. In addition, overlapping functions of various chemokine–chemokine receptor pairs contribute, as seen by the competing relevance of CXCR3 expression on donor T cells for the development of hepatic and gastrointestinal GVHD .
Chemokine receptor:ligand interactions have been previously shown to modulate alloreactive T cell responses . In our study, treatment of T cells with NR58-3.14.3 in vitro resulted in decreased T cell activation and proliferation in response to alloantigen presenting cells (APCs) but not to mitogens. It is likely that NR58-3.14.3 alters the effects of APC-produced chemokines on responder T cells by interfering with T cell signaling, and hereby not only changes the migratory but also the proliferative capacity of these cells.
While NR58-3.14.3 reduced alloreactive T cell proliferation in vitro, it did not suffice to block T cell proliferation in vivo. Plasma half life time of NR58-3.14.3 is short, and clearance in vivo is renal with essentially no metabolism [41, 42]. If all the panoply of in vivo metabolism systems do not significantly metabolise this reagent, it is highly likely that NR58-3.14.3 is stable in in vitro cultures. In vivo steady state levels in mice achieved by 30 mg/kg body weight daily via an implantable osmotic minipump have been reported with 10–15 μM . However, subcutaneous application in vivo leads to high spike concentrations, followed by a period when drug levels are undetectable due to its pharmacokinetics , and it cannot be excluded that differences in drug levels obtained in vivo compared to drug levels present in cell cultures in vitro led to the observed discrepancy of NR58-3.14.3 treatment on alloantigen-specific T cell expansion and activation in vitro and in vivo.
As shown by histopathology, the chosen application scheme for NR58-3.14.3 in this study efficiently decreased the evasion of donor effector cells into certain, although not all target tissues. These heterogeneous effects of preventive NR58-3.14.3 treatment on organ-specific GVHD may be due to different tissue levels of BSCI in different GVHD target organs or may be related to the increased organ expression of different members of the chemokine network.
Treatment with NR58-3.14.3 was associated with a minor benefit on clinical GVHD, which most likely can be directly attributed to the reduced severity of hepatic and pulmonary injury, as no differences were found for systemic inflammation and neither did the minor but statistically significant reduction in T cell proliferation in vitro translate into decreased T cell expansion in the complex setting of immune activation and inflammation after allo-BMT in vivo.
In conclusion, our results show that preventive broad spectrum chemokine inhibition using NR58-3.14.3 ameliorates the severity of pulmonary and hepatic GVHD. However, beneficial effects of NR58-3.14.3 do not apply to all GVHD target organs, and as seen for other immunosuppressive approaches, treatment with BSCI may lead to increased susceptibility to infection, which has to be tested in future studies. The development and use of novel drugs such as NR58-3.14.3 targeting multiple chemokines or their receptors will be important steps to better prevent and treat progressive lung injury and GVHD after allo-BMT in the near future.
We would like to thank Funxional Therapeutics (Cambridge, UK) for generously providing NR58-3.14.3. DJG is a Director and acts as consultant CSO for Funxional Therapeutics Ltd (Cambridge, UK), who own and develop BSCIs for various indications, with an initial focus on asthma. GCH is a Max Eder Research Fellow of the Deutsche Krebshilfe e.V. and this work was supported by the German Cancer Foundation (Deutsche Krebshilfe e.V.), Project #106647.