The chemokine CXCL13 is a key regulator of B cell recruitment to the cerebrospinal fluid in acute Lyme neuroborreliosis

Abstract

Background: The chemokine CXCL13 is known to dictate homing and motility of B cells in lymphoid tissue and has been implicated in the formation of ectopic lymphoid tissue in chronic inflammation. Whether it influences B cell trafficking during acute infection, is largely unclear. In previous studies, we showed that (I) CXCL13 levels are markedly increased in the B cell-rich cerebrospinal fluid (CSF) of patients with acute Lyme neuroborreliosis (LNB), and (II) CXCL13 is released by monocytes upon recognition of borrelial outer surface proteins by Toll-like receptor 2. Here, we assessed the role of CXCL13--in comparison to other chemokines--in the recruitment of B cells to the CSF of patients with acute LNB.

Methods: Measurement of chemokines was done by ELISA. B cells were isolated from whole blood using magnetic cell separation (MACS). For migration experiments, a modified Boyden chamber assay was used and the migrated B cells were further analysed by FACS. The migration was inhibited either by preincubation of the CSF samples with neutralizing antibodies, heating to 60 degrees C, removal of proteins >3 kDa, or by pre-treatment of the B cells with pertussis toxin. The principal statistical tests used were one-way analysis of variance and Bonferroni test (chemokine measurements) as well as paired Student's t-test (migration experiments).

Results: Measurements of chemokine levels revealed an increase in three of the four known major B cell chemoattractants CXCL13, CCL19 and CXCL12 in LNB CSF. The CXCL13 CSF:serum ratio, as a measure of the chemotactic gradient, was substantially higher than that of CCL19 and CXCL12. Moreover, the chemotactic activity of LNB CSF was reduced up to 56% after preincubation with a neutralizing CXCL13 antibody, while combined preincubation with antibodies against CXCL13, CCL19, and CXCL12 did not lead to further reduction. Since treatment with pertussis toxin, heating to 60 degrees C, and removal of proteins >3 kDa abrogated the chemotactic activity, further not yet identified chemokines seem to be involved in B cell recruitment to LNB CSF.

Conclusion: Combined, our study suggests a key role of CXCL13 in B cell migration to sites of infection as shown here for the CSF of LNB patients.

Figure 1

Migration of B cells to CSF samples. (A) migration index of CD19⁺ B cells to CSF samples from different patient groups, horizontal bars represent the mean value, *: p < 0.001. (B) and (C) exemplary result of a FACS analysis of (B) migrated or (C) not-migrated CD19⁺CD27⁺ B cells towards the CSF sample of a LNB patient. (D) percentage of migrated vs. not-migrated CD27⁺CD19⁺ B cells, using either CSF-samples of patients with LNB (n = 3) or 500 ng/ml rhCXCL13 (mean of two independent experiments, done in duplicate) as chemotactic agents.

Figure 2

Concentration of Chemokines in CSF samples. Concentration of the respective chemokines in either CSF- (A, D, and G) or serum-samples (B, E, and H) of the different patient groups, and accordingly the calculated CSF-to-serum ratio (C, F, and I), horizontal bars represent the mean value, *: p < 0.001, $: p = 0.003, and #: p = 0.021 vs. LNB, and +: p < 0.001, and § p = 0.013 vs. NS

Figure 3

Correlation of the CSF-to-serum ratio of chemokines with the migration index. Correlation of the CSR of the different chemokines with the MI of the respective CSF sample as measured in the migration assay. A cut-off for the MI was set at 80, as all CSF samples from NIND patients (and accordingly no inflammatory cell infiltrate in the CSF) had a MI below this value (horizontal dotted line). The vertical dotted line separates the samples with a "negative" CSR (<1) from those with a "positive" CSR (>1). A high proportion of CSF samples in the upper right or lower left quadrant indicate a positive association.

Figure 4

Inhibition of migration with neutralizing antibodies. (A) Inhibition of the chemotactic effect of CSF samples from 9 LNB patients (same enumeration as in table 1) with the indicated antibody (in % compared with the respective isotype control). (B) All antibodies were used separately in patient Nr. 3, where the CXCL13 antibody alone had no relevant effect.

Figure 5

Inhibition of migration with PTx, heating, or filtration of substances >3 kDa. CSF samples of LNB patients (n = 3, black bars) or 500 ng/ml rhCXCL13 (mean of two independent experiments, done in duplicates, gray bars) were used as chemotactic agent. Either, the B-cells were preincubated with PTx, or the chemotactic agent was either filtrated to eliminate substances larger than 3 kDa or heated at 60°C prior to the migration assay. The reduction of migration was compared to the untreated B cells, or the untreated chemotactic agents, respectively.