CXCR4/CXCL12 Hyperexpression Plays a Pivotal Role in the Pathogenesis of Lupus

Mice

Male B6, B6.Sle1, B6.Yaa, B6.Sle1Yaa, BXSB, and MRL.lpr mice and female B6.Sle1Sle2Sle3 mice were purchased from The Jackson Laboratory or bred in our colony at the University of Texas Southwestern Medical Center and housed in the University of Texas Southwestern Medical Center Animal Resources Center’s specific pathogen-free facility. The care and use of laboratory animals in our facility conforms to the National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved animal protocols.

Flow cytometry and Abs

Abs to the following mouse Ags were used for flow cytometry: CD21-FITC, CD23-PE, IgM-PerCP-Cy5.5, IgM-PE-Cy7, CD5-allophycocyanin, B220-allophycocyanin-Cy7, B220-FITC, CXCR4-PE, CXCR4-biotin, GL7-FITC, CD138-PE, CD69-PE, CD69-PerCP-Cy5.5, Gr-1-allophycocyanin-Cy7, CD3-FITC, CD4-allophycocyanin-Cy7, CD86-PE, AA4.1-FITC, CD43-PE, CD45-FITC, CD45-PE-Cy7, NK1.1-PE-Cy7, annexinV-PE (BD Biosciences); CD11b-allophycocyanin, CD8-PE-Cy7, CD44-allophycocyanin, CD11c-Pacific Blue, Ie/Ib-FITC, F4/80-PE-Cy5, CD3-Pacific Blue (eBioscience); and CD19-PE-Texas Red, CD62L-PE-Texas Red, PDCA-1-biotin, strepavidin-Qdot 655, strepavidin-PE-Texas Red, and 7-aminoactinomycin D (Invitrogen). Samples were Fc-blocked with the 2.4G2 Ab or 10% normal rabbit sera (Invitrogen). For spleen, bone marrow, and blood, at least 5 × 10⁴ cells were acquired in the live gate, as defined by size and granularity. For kidney samples, at least 5 × 10⁵ cells were acquired on the leukocyte gate, as defined by size and CD45 positivity. Samples were either acquired on an LSR II flow cytometer or FACSCalibur (BD Biosciences) and analyzed using FlowJo (Tree Star). MFIs represent mean fluorescent intensities.³

Isolation of renal leukocytes

Mice were perfused with 20 ml of cold PBS to remove the blood (Invitrogen). One-half of a kidney was harvested for histological analyses. The remainder was mechanically disrupted and incubated with 1 mg/ml collagenase IV (Sigma-Aldrich) and 1000 U/ml DNase (Roche) for 5 min in a 37°C water bath, and then at 37°C for 30 min with shaking. Cells were furthered mechanically disrupted by passage through a 23-gauge needle and then subjected to hypotonic shock with potassium acetate lysis buffer to remove residual RBC (Sigma-Aldrich). Cells were centrifuged at 3000 rpm at 25°C through a 40% Percoll (Sigma-Aldrich) gradient for 20 min. Leukocytes were recovered from the pellet and then enumerated by trypan blue exclusion and subjected to flow cytometric analysis.

In vitro splenocyte stimulation

Sterile splenocyte suspensions were plated in triplicate at 0.5 × 10⁶ cells/ml in complete medium (RPMI 1640 with l-glutamine, 10% FCS (HyClone), HEPES (Sigma-Aldrich), 1× penicillin/streptomycin (Sigma-Aldrich), 2-ME (Sigma-Aldrich), l-glutamine (Sigma-Aldrich)) with varying concentrations of the following stimuli: recombinant CXCL12 (R&D Systems), with or without anti-IgM F(ab′)₂, and anti-CD40 (Jackson ImmunoResearch Laboratories). Cells were cultured at 37°C for 72 h and analyzed by flow cytometry.

In vitro macrophage stimulation

Bone marrow-derived macrophages were derived from 6- to 8-wk-old B6 males, as described (31). Macrophages were plated at 5 × 10⁵ cells/ml and stimulated for 24 h with TNF-α (0.05 ng/ml), IL-1-β (10 pg/ml), and/or IL-6 (0.1 ng/ml) (all from R&D Systems). For the blocking studies, macrophages were stimulated with 10 ng/ml LPS (Sigma-Aldrich) and treated with anti-TNF-α (0.5 μg/ml), anti-IL-1β (5 μg/ml), and/or anti-IL-6 (0.5 μg/ml) (all from R&D Systems). For flow cytometry analyses, at least 3 × 10⁴ cells were acquired in the macrophage gate (i.e., cells that were CD11b⁺F4/80⁺) and examined for CXCR4 expression.

Transwell migration

Transwell plates (6.5 mm) with 5.0-μm-pore polycarbonate membranes (Corning) were used. Six hundred microliters of 50 ng/ml CXCL12 was placed in the lower chamber and preincubated at 37°C for 2 h to equilibrate the membrane. Splenocytes (1.5 × 10⁶) were loaded into the upper well of the transwell plate. In some experiments, cells were resuspended in complete RPMI 1640 medium containing various doses of the CXCR4 inhibitor CTCE-9908 (Chemokine Therapeutics). Cells were allowed to migrate for 2 h at 37°C. Cells in the lower chamber were counted using trypan blue exclusion and analyzed by flow cytometry.

Renal cell supernatant and ELISA

Mice were perfused with 20 ml of PBS, and single-cell suspensions of the kidney were prepared by mechanical disruption and collagenase digestion. Cell suspensions (500 μl of PBS per two kidneys) were centrifuged at 1,500 rpm for 7 min, and the supernatant was collected and further centrifuged at 14,000 rpm for 10 min. Supernatant (termed “renal plasma”) was collected and used at various dilutions to quantitate CXCL12 concentration by ELISA (R&D Systems). OD₄₅₀ was measured by an Elx800 automated microplate reader (BioTek Instruments) and concentrations were extrapolated from a 4-point standard curve (R² > 0.99), where the mean of experimental duplicates was used.

Immunohistochemistry

Paraffin sections (5 μm) of the kidney were boiled in 10 mM citrate, quenched with 0.01% NaBH₄, blocked with murine F(ab′)₂ anti-IgG (Jackson ImmunoResearch Laboratories), and stained with anti-mouse SDF-1/CXCL12 Ab (R&D Systems) at 4°C overnight. Sections were then washed and incubated with goat anti-mouse IgG conjugated to Cy3 (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Sections were washed and then mounted with Vectashield (Vector Laboratories), visualized, and photographed with a Zeiss Axioplan 2 digital camera and analyzed using AxioVision (Zeiss).

In vivo inhibition of CXCR4

The peptide antagonist CTCE-9908 (Chemokine Therapeutics) was resuspended in 5% dextrose water. Two cohorts of B6.Sle1Yaa male mice were assembled for the in vivo “prevention” or “treatment” studies. In cohort I, comprised of 2-mo-old mice, 5 were injected with vehicle and 11 received CTCE-9908. In cohort II, comprised of antinuclear autoantibody (ANA) -seropositive 4-mo-old mice, 5 were injected with vehicle and 8 were injected with CTCE-9908. In both study groups, 100 μl of CTCE-9908 at 50 mg/kg or vehicle placebo were injected i.p. three times a week for the course of study (i.e., until 6 mo of age or until death). All mice were monitored for serum autoantibodies, proteinuria, azotemia, and evidence of renal pathology, as detailed below.

Serology

Mice were bled before and at 1, 2, 3, and 4 mo after treatment with CTCE-9908 or placebo and the sera were stored at −20°C. ELISA detection of serum IgM and IgG autoantibodies to chromatin and dsDNA were performed as described (13). OD₄₅₀ was measured using an Elx800 automated microplate reader (Bio-Tek Instruments), and the raw optical densities for anti-chromatin Abs were converted to arbitrary normalized units using a 6-point standard curve generated by an antinuclear mAb derived from a NZM2410 mouse (13).

Assessment of renal disease

Twenty-four hour urine samples were collected using metabolic cages for proteinuria analyses, which were assessed using the Coomassie Plus protein assay kit (Pierce) with BSA as a standard. OD₆₃₀ was measured using an Elx800 automated microplate reader (Bio-Tek Instruments). Blood urea nitrogen was assessed using the QuantiChrom urea assay kit (BioAssay Systems). The extent of glomerular and tubulointerstitial disease and the percentage of glomerular crescent formation were scored as detailed elsewhere (14).

Statistical analysis

For the in vivo disease prevention/treatment studies, the experimental mice were compared with the placebo group wherever sufficient placebo mice were still alive. For the 6 mo of age comparisons, since most of the placebo controls were already dead, 5- to 6-mo-old untreated B6.Sle1Yaa mice were used as controls for the statistical analyses. Data were analyzed using InStat3 or GraphPad 4 (GraphPad Software). Where appropriate, one-way ANOVA with Dunnett or Bonferroni post hoc analysis, Welch t test, Mann-Whitney U test, or log rank (Mantel-Cox) test were used. Error bars represent SEMs.

Increased CXCR4 expression on leukocytes of multiple murine lupus strains is a consequence of disease

Examination of several murine lupus models, including BXSB, MRL.lpr, and B6.Sle1Yaa, revealed that CXCR4 was significantly up-regulated on multiple cell types in 8- to 10-mo-old mice, regardless of their genetic composition, when compared with B6 controls (Fig. 1⇓ and Table I⇓). Similar differences were noted in a fourth lupus-prone strain, B6.Sle1Sle2Sle3 (Table I⇓). Cell types that exhibited the largest up-regulation of CXCR4 compared with B6 in all murine models included cell subsets of both the myeloid and B cell lineages. In particular, inflammatory monocytes, neutrophils, plasma cells, and pre-plasma cells expressed the highest absolute levels of CXCR4, particularly in the lupus-prone strains (Fig. 1⇓ and Table I⇓). Both in terms of the MFIs, as well as the percentage of cells expressing CXCR4, myeloid and B cells from lupus mice exhibited an ∼2-fold increase in surface expression of CXCR4. Although these differences were most marked on splenic leukocytes, a similar pattern of expression difference was also noted on leukocytes isolated from lymph nodes, peripheral blood, and the bone marrow (Table I⇓ and data not shown). Although we detected an increase in CXCR4 expression on memory CD4⁺ T cells, these results did not reach statistical significance.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Increased CXCR4 expression in murine models of lupus. Splenic (A) and peripheral blood (B) leukocytes from 9- to 12-mo-old male B6.Sle1Yaa, BXSB, MRL.lpr, and B6 mice (n = 6–10 each) were subjected to flow cytometric analyses. Increased expression of CXCR4 was detected on neutrophils (N0, CD11b⁺, Gr-1^high), inflammatory monocytes (Inflammatory M0, CD11b⁺, Gr-1^int), and plasma cells (B220^low, CD138⁺) in the spleen and peripheral blood, compared with B6. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; by Welch-corrected t test. The MFIs and the percentage of each cell type that expressed CXCR4 are detailed in Table I. Shown in C are representative histogram overlays of CXCR4 staining.

CXCR4 is located on murine chromosome 1 at 130 Mb, centromeric to the Sle1 interval (with Sle1b being located at 174 Mb). To examine if CXCR4 hyperexpression was due to a genetic polymorphism or whether it arose as a consequence of disease, we adopted two approaches, using the B6.Sle1Yaa strain as a disease model. First, evaluation of young (2-mo-old) predisease B6.Sle1Yaa mice did not reveal a similar degree of CXCR4 hyperexpression compared with young B6 controls (Fig. 2⇓A). Second, we examined monocongenic mice bearing the two main genetic elements that dictate lupus development in B6.Sle1Yaa mice: Sle1^z and Yaa (15, 16, 17, 18). Interestingly, the up-regulation of CXCR4 was present only in the bicongenic mice, and only in mice with active lupus, defined by the presence of IgG ANA (antichromatin) seropositivity and active proteinuria >1 mg/24 h (Fig. 2⇓). In contrast, the up-regulation of CXCR4 was not observed in B6.Sle1 or in B6.Yaa mice (Fig. 2⇓). Furthermore, direct sequencing of the CXCR4 gene revealed no sequence polymorphisms between B6 and B6.Sle1Yaa (data not shown). Taken together, these data indicate that the up-regulation of CXCR4 on B6.Sle1Yaa leukocytes is unlikely to be the direct consequence of any single genetic contribution responsible for disease (i.e., Sle1 or Yaa), but is instead likely to be the downstream consequence of the disease process.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

CXCR4 hyperexpression in B6.Sle1Yaa mice is not the direct consequence of either locus, but arises as a result of the disease. A, Comparison of mean percentage of CXCR4⁺ cells on young (age ≤2 mo) B6.Sle1Yaa mice compared with age-matched B6 mice (n = 15). B and C, Mean percentages of myeloid cells or plasma cells that express CXCR4 in 9-mo-old B6.Sle1Yaa mice or the related age-matched monocongenic strains (n = 6 each) in the blood and spleen, respectively. ANOVA with Dunnett posthoc test was used for statistical analyses: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

TLR stimulation and inflammatory cytokines may be responsible for the increased CXCR4 expression in murine lupus

We next explored the mechanistic basis of CXCR4 up-regulation in lupus. B cell receptor ligation with or without CD40L ligation or PMA was not able to up-regulate CXCR4 on B cells; likewise, stimulation of T cells in vitro with anti-CD3, anti-CD28, PMA, or Con A were also not able to up-regulate CXCR4 (data not shown). In contrast, TLR ligation and inflammatory cytokines appeared to be potent at up-regulating CXCR4 expression in vitro (Fig. 3⇓A). This was partly dependent on inflammatory cytokine production since LPS-induced CXCR4 up-regulation was partly dampened by Abs to IL-1β, Il-6, and TNF-α (Fig. 3⇓A). Interestingly, in all conditions in which IL-6 was neutralized, we observed a significant decrease in CXCR4 expression compared with LPS-treated macrophages, although both direct signaling through TLR4 as well as synergistic effects of both IL-1-β and TNF-α seem to also play a role in mediating CXCR4 up-regulation. Likewise, when we stimulated macrophages directly with these recombinant cytokines, all conditions in which IL-6 was added led to significantly increased CXCR4 expression compared with unstimulated macrophages (Fig. 3⇓B). These observations are likely to be physiologically relevant since these cytokines were elevated in murine lupus, including the B6.Sle1Yaa strain (Fig. 3⇓, C–E) that shows prominent CXCR4 up-regulation on various leukocyte subsets. Collectively, these studies indicate that TLR ligation and inflammatory cytokines (rather than Ag receptor stimulation) may be playing key roles in orchestrating the CXCR4 increase seen in lupus. On the other hand, neither stimulation of cells with the TLR7 agonist R837, the cytokine IFN-α, or a variety of TLR9 agonistic CpG oligodeoxynucleotides were able to up-regulate CXCR4 (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Increased CXCR4 expression in B6.Sle1Yaa mice is mediated by TLR ligation and inflammatory cytokines. A, Expression of CXCR4 on B6 bone marrow-derived macrophages following LPS stimulation in the presence of neutralizing Ab to IL-6, TNF-α, and IL-1-β. Two mice did not respond to LPS under any condition and were omitted from the graph. Statistics pertain to differences in mean CXCR4 expression relative to LPS-treated macrophages. B, Expression of CXCR4 on B6 bone marrow-derived macrophages following stimulation with exogenous IL-6, TNF-α, and/or IL-1β. Statistics pertain to differences in mean CXCR4 expression relative to untreated macrophages. C–E, Serum cytokine concentrations of IL-6, TNF-α, and IL-1β in B6.Sle1Yaa and B6 mice at their indicated ages. ANOVA with Dunnett post hoc test was used for all statistical analyses: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Increased CXCR4 expression leads to increased migration to positive CXCL12 gradients and increased B cell survival

We next examined the functional consequences of up-regulation of CXCR4 in diseased mice. Three-fold more B6.Sle1Yaa splenocytes, which express more CXCR4, migrated to positive CXCL12 gradients when compared with B6 controls (Fig. 4⇓A). Among the cell types that expressed CXCR4, the migratory potential of neutrophils, monocytes, and B cells differed most significantly between the strains, although all cell types from diseased mice that express higher CXCR4 levels migrated better to CXCL12 compared with the B6 controls (Fig. 4⇓ and data not shown). Also, when CXCL12 was added to both the top and bottom chambers, no significant migration was observed (data not plotted), excluding chemokinesis as a possible explanation for these data.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Functional consequence of CXCR4 hyperexpression on B6.Sle1Yaa leukocytes. A, Splenocytes from 9- to 12-mo-old B6, B6.Sle1, B6.Yaa, and B6.Sle1Yaa mice were subjected to transwell migration assays toward 50 ng/ml CXCL12 placed in the bottom chamber. Cells that had extravasated into the bottom chamber were enumerated and phenotyped by flow cytometric analyses. Representative flow plots are shown on the right. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 by ANOVA with Dunnett post hoc analyses. B, B6 and B6.Sle1Yaa splenic B cells were stimulated with anti-IgM F(ab′)₂ Abs and recombinant CXCL12 and assayed by flow cytometry for apoptosis, using annexin V and 7-aminoactinomycin D. Representative flow plots are shown on the right.

In addition to its role in chemotaxis, CXCL12 has also been shown to impact B cell lymphopoiesis (19). Therefore, we assessed whether increased CXCR4 expression affected the responses of B cells to stimulation with CXCL12. We found that BCR-ligated B cells from B6.Sle1Yaa spleens showed significantly better survival when exposed to CXCL12 (Fig. 4⇑B). We detected no differential B cell responses to CXCL12 in terms of proliferation or the expression of activation markers assessed (data not shown). We also detected no difference in the proliferation or activation of B6.Sle1Yaa T cells when exposed to CXCL12 (data not shown). These data indicate that the increased expression of CXCR4 on lupus leukocytes is associated with multiple functional consequences, the most profound of which include enhanced BCR-triggered B cell survival and chemotaxis to positive CXCL12 gradients.

Increased renal expression of CXCL12 in lymphoproliferative lupus

We next examined if the increased renal disease in lupus might be in part driven by heightened CXCR4/CXCL12 activity. We observed a robust increase in CXCL12 expression in the kidneys of B6.Sle1Yaa, but not B6, mice both by immunohistochemistry (Fig. 5⇓A) and by ELISA (Fig. 5⇓B). We observed increased CXCL12 expression in both the glomeruli and the tubules. More CXCL12 was also expressed in the interstitium, as well as in the Bowman’s capsules of B6.Sle1Yaa kidneys, compared with B6 controls (Fig. 5⇓A). Similar increases were also observed in BXSB and MRL.lpr kidneys (data not shown). In addition to the increase in CXCL12, we also noted CXCR4⁺ cells to accumulate within B6.Sle1Yaa kidneys when examined ex vivo (Fig. 6⇓). Collectively, these data suggest that the CXCR4/CXCL12 axis may be instrumental for leukocyte trafficking into the kidneys, and thus it may play an important role in mediating renal pathology in lupus nephritis.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

Increased CXCL12 expression in lupus kidneys. A, CXCL12 was detected on paraffin-fixed sections of 9- to 12-mo-old B6.Sle1Yaa kidneys (and B6 controls) using immnohistochemistry. Displayed below are the zoom-in magnifications of the same images. Images shown are representative of five independent samples for each strain. B, Renal plasma harvested (as described in Materials and Methods) from 9- to 12-mo-old B6 and B6.Sle1Yaa mice (n = 6 each) were examined for CXCL12 by ELISA. ∗∗, p < 0.01 by Welch-corrected t test.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Accumulation of CXCR4⁺ leukocytes in lupus kidneys. Whole kidneys from the indicated strains (at 9–12 mo of age) were subjected to digestion and Percoll density centrifugation, as detailed in Materials and Methods. The fraction enriched for leukocytes was enumerated using trypan blue exclusion and subjected to flow cytometric analyses. A, Cellular constituents of the leukocyte-enriched preparations. The SSC/CD45-defined gate depicted in the top row was used to identify leukocytes, which were then characterized using additional surface markers, as shown in the second and third rows. B, Numbers of CXCR4⁺ cells (left) and the percentage of each cell subset that expressed CXCR4 (right) in B6.Sle1Yaa cells and cells from the relevant monocongenic strains and B6 control. Data shown are representative of three mice per group. Neutrophils were defined as CD45⁺, NK1.1⁻, CD11b⁺, Gr-1^high, SSc^high; inflammatory monocytes were defined as CD45⁺, NK1.1⁻, CD11b⁺, Gr-1^int; and resident monocytes were defined as CD45⁺, NK1.1⁻, CD11b⁺, Gr-1⁻. Shown p values pertain to comparisons of the B6.Sle1Yaa levels against those of B6.Sle1 and B6.Yaa by ANOVA (Bonferroni post hoc test): ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Pharmacological inhibition of CXCR4 significantly ameliorates disease

To explore if increased CXCR4/CXCL12 might be a good therapeutic target in lupus, we tested the CXCR4 peptide antagonist CTCE-9908. Initial characterization indicated that CTCE-9908 was effective in blocking chemotaxis by B6.Sle1Yaa splenocytes in vitro, with the migration of all cell types being inhibited equally (supplemental Fig. S1).⁴ To test the therapeutic efficacy in vivo, B6.Sle1Yaa mice were subjected to two sets of placebo control studies. First, the preventive efficacy of CXCR4 blockade was tested on a cohort of 2-mo-old B6.Sle1Yaa mice (cohort I), which typically do not have antinuclear Abs or nephritis. Second, the therapeutic efficacy of CXCR4 blockade was tested on 4-mo-old B6.Sle1Yaa mice (cohort II), which by this age have already developed detectable impairment of renal function, high titers of ANAs, and a large spectrum of immunological changes (18).

Blocking the CXCR4 axis from the age of 2 mo significantly prolonged survival (by about 2 mo) and reduced splenomegaly (p < 0.05), T cell and B cell activation (p < 0.05), autoantibody production (p < 0.001), and nephritis (p < 0.01) (Fig. 7⇓ and Table II⇓). In particular, tubulointerstitial disease and crescent formation, rather than glomerulonephritis, were the pathological features that were most significantly reduced (p < 0.01; Fig. 7⇓). The observed change in splenomegaly was accompanied by significant reductions in peripheral T and B cells, as well as various myeloid cells (cohort I in Table II⇓). Importantly, similar disease amelioration and lifespan prolongation was also seen when treatment was started after disease onset (Fig. 8⇓ and data not shown). Although splenic cellularity was not reversed in the latter study (cohort II in Table II⇓ and data not shown), the treatment regimen significantly reduced renal disease, including tubulointerstitial disease (p < 0.01), glomerulonephritis (p < 0.001), and glomerular crescent formation (p < 0.05) (Fig. 8⇓). The absolute numbers of leukocytes recruited into the kidneys, particularly various myeloid cell subsets, were significantly reduced by CXCR4 blockade, both in the prevention (p < 0.001) and treatment (p < 0.01) studies (Table II⇓ and Fig. 9⇓). Both the preventation and therapeutic treatments were not associated with any changes in body weight, peripheral RBC counts, hemoglobin levels, or liver function tests (data not shown), alluding to the safety of the administered drug.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Early administration of a CXCR4 peptide antagonist prevents lupus. Two-month-old B6.Sle1Yaa males (cohort I) were injected with CTCE-9908 (n = 11) or vehicle placebo (n = 5). All mice were sacrificed at the age of 5–6 mo. One hundred microliters of CTCE-9908 at 50 mg/kg or vehicle placebo was injected i.p. three times a week for the course of study (i.e., from the age of 2 mo until 5–6 mo of age or until spontaneous death). A, Survival curve for mice in the prevention study (cohort I). B, Spleen sizes upon sacrifice of the mice; examples of spleens from the respective strains are presented as insets. C, Antichromatin ANA levels (as assayed by ELISA) over the course of the treatment regime. D, Total proteinuria levels detected in 24 h urine collections at the indicated ages. E, Histopathological findings upon sacrifice at 6 mo. CTCE-9908 and Placebo refer to B6.Sle1Yaa mice that received experimental or placebo treatment, respectively, while B6 and B6.Sle1Yaa refer to unmanipulated controls (n = 5 each). Top, Representative histological images: 1, B6 control; 2, B6.Sle1Yaa; 3, CTCE-9908 treated; 4, placebo-treated. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 by log rank (Mantel-Cox) test, Welch-corrected t test, or the Mann-Whitney U test, where appropriate. Note that for all studies executed at the terminal 6-mo age point (B–E), untreated 5- to 6-mo-old B6.Sle1.Yaa mice were used as the control group because insufficient placebo-treated mice survived to 6 mo of age (as can be surmised from A).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 8.

CXCR4 peptide antagonist decreases the severity of lupus nephritis even when administered after disease onset. Four-month-old ANA-seropositive B6.Sle1Yaa males (cohort II) were injected with CTCE-9908 (n = 8) or vehicle placebo (n = 5). One hundred microliters of CTCE-9908 at 50 mg/kg or vehicle placebo was injected i.p. three times a week for the course of study (i.e., from the age of 4–6 mo or until spontaneous death). A, Survival curve for mice in the treatment study (cohort II). B, Total proteinuria levels detected in 24-h urine collections at the indicated ages. C, Glomerulonephritis scores, tubulointerstitial disease scores, and extent of glomerular crescent formation in kidneys obtained from the different groups of mice studied, upon sacrifice at 6 mo. CTCE-9908 and Placebo refer to B6.Sle1Yaa mice that received experimental or placebo treatment, respectively, while B6 and B6.Sle1Yaa refer to unmanipulated controls (n = 5 each). Right, Representative histological images: 1, B6 control; 2, B6.Sle1Yaa; 3, CTCE-9908 treated; 4, placebo treated. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 by log rank (Mantel-Cox) test, Welch-corrected t test, or the Mann-Whitney U test, where appropriate. Note that for all studies executed at the terminal 6-mo age point (B and C), untreated 5- to 6-mo-old B6.Sle1.Yaa mice were used as the control group because insufficient placebo-treated mice survived to 6 mo of age (as can be surmised from A).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 9.

CXCR4 blockade reduces renal leukocyte infiltration in murine lupus nephritis. Two cohorts of B6.Sle1Yaa male mice were subjected to prevention or treatment studies using the CXCR4 peptide antagonist, CTCE-9908, as detailed above in Figs. 7 and 8. A, Absolute numbers of CD45⁺ cells in the kidneys of the indicated mice upon sacrifice at 6 mo; indicated below are representative two-dimensional plots demonstrating reduced percentage of leukocytes in the renal cell preparation. B, Gr-1/CD11b staining profiles of myeloid cell subsets within the kidneys in the different groups of mice upon sacrifice at 6 mo, and the absolute numbers of various myeloid cell subsets in the kidneys of mice from the different study groups, as further detailed in Table II (n = 4–6 mice/group). Neutrophils were defined as CD45⁺, NK1.1⁻, CD11b⁺, Gr-1^high, SSc^high; inflammatory monocytes were defined as CD45⁺, NK1.1⁻, CD11b⁺, Gr-1^int; and resident monocytes were defined as CD45⁺, NK1.1⁻, CD11b⁺, Gr-1⁻. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 by log rank (Mantel-Cox) test, Welch-corrected t test, or the Mann-Whitney U test, where appropriate.