Mechanisms and functions of lymphangiogenesis in regulating the immune response and inflammation resolution 2

Our data supported a scenario whereby interplay between LN interstitial flow, fibroblastic reticular network and spatial-temporal distribution of VEGF-A formed coordinating forces to dri

Figure 5.1 Spatial differences in VEGF-A distribution accompany the differential remodeling of cortical and medullary sinuses during prolonged inflammation (A) VEGF-A expression was largely confined to the subcapsular B cell

regions of the activated LNs at day 4 post-immunization but was also detected within the T cell zone and LN medulla at day 14 Scale bar in represents 400 µm (B) Confocal images revealed that while VEGF-A co-localized with subcapsular sinuses

on both day 4 and 14 after immunization, VEGF-A co-localization with the cortical and medullary sinuses were detected only on day 14 Scale bars represent 50µm

Given the intricate relationship between FRC network and lymphatic channels, we also examined VEGF-A distribution with respect to the ER-TR7+ reticular fibers associated with lymphatics VEGF-A co-localized with the reticular network that lined subcapsular sinuses (identified by presence of DCs within lymphatics in previous sequential section, not shown) (dotted white lines demarcated subcapsular lymphatics) on both day 4 and 14 post-immunization (Fig 5.2A) Interestingly, while VEGF-A was largely absent from the ER-TR7+ network associated with cortical and medullary sinuses (identified by absence of DCs in lumen of LYVE-1+ sinuses in previous sequential section, not shown) (dotted while lines demarcate cortical and medullary sinuses, lumen marked by L) on day 4 post-immunization, there was an obvious association of VEGF-A with the ER-TR7+ FRC fibers that lined cortical and medullary sinuses at day 14 post-immunization (Fig 5.2A) Association between FRCs lining cortical and medullary sinuses and VEGF-A was ascertained to occur at

2 interfaces - VEGF-A was present at the surface and inside reticular fibers (Fig 5.2B) While the observation of VEGF-A within and on the reticular fibers is suggestive that VEGF-A is produced by FRCs and secreted into the LN parenchyma,

we cannot exclude the possibility that extra-nodal VEGF-A may be transported within reticular conduits and subsequently displayed on FRCs

To further explore the relationship between lymphatics and FRCs, we used VEGFR3

as another marker for lymphatics We observed similar close spatial association between lymphatics and FRCs (Fig 5.2C) Closer examination also revealed that cortical and medullary sinuses, FRCs and VEGF-A in LNs from day 14 post-immunization engaged in a tripartite interaction whereas such an interaction was not observed at day 4 (Fig 5.2C)

Altogether, these data indicate that as the inflammation evolved, spatial differences in the distribution of VEGF-A within DLNs may mediate the remodeling of cortical and medullary sinuses

Figure 5.2 Association of VEGF-A with the FRCs lining lymphatics (A) VEGF-A

co-localized with FRCs that lined subcapsular sinuses on both day 4 and 14 after immunization VEGF-A association with FRCs that lined cortical and medullary sinuses was detected only on day 14 post-immunization Dotted while lines demarcates lymphatics (B) Orthogonal plane view of how VEGF-A is aligned on the FRCs lining cortical and medullary sinuses Inset shows enlarged image of confocal image stack of boxed region in E VEGF-A can be found on the surface of as well as inside FRCs; (C) The interaction between cortical-medullary lymphatics, FRCs and VEGF-A in LNs on day 14 post-immunization Scale bars represent 50µm L = lumen

5.2.2 Interstitial flow is required for the differential distribution of VEGF-A in lymph node during inflammation

Interstitial flow acting in concert with lymphangiogenic factor has been reported to be

a key driving force of lymphangiogenesis (Boardman and Swartz, 2003; Goldman et al., 2007) Moreover, alterations in interstitial flow have also been shown to be important for the expression of chemokines by FRCs (Tomei et al., 2009) We therefore considered the possibility that interstitial flow through the LNs during inflammation might influence the expression and distribution of VEGF-A and, thereby support differential LV remodeling To address this, we designed a surgical strategy to perturb LN interstitial flow by cutting the afferent lymphatics draining the popliteal LN at day 10 post-immunization Mice that received sham operations served

as controls (Fig 5.3A) Mice were sacrificed 1 day after surgery Patency or obstruction of lymphatic flow to the popliteal LN was verified (Fig 5.3B)

Figure 5.3 LV surgery to disrupt lymph flow to the popliteal lymph node (A)

Afferent lymphatic vessels were cut on one side (‘LV resection’) while a sham operation was performed on LVs on the contralateral side (‘sham’) FITC-dextran uptake was used as a marker of lymphatic transport and flow LV transport of FITC- dextran to LN following ‘LV resection’ was cut off compared to the ‘sham’ LN (B)

At sacrifice, FITC-dextran injected into footpads of mice verified that lymph flow to the popliteal LNs was still functional in the ‘sham’ side and not patent in the side with

‘LV resection’ The popliteal LN with the intact LV was brightly fluorescent while fluorescence of the popliteal LN drained by the severed LV was dim Scale bar in (B) represents 2mm

While VEGF-A expression was preserved in the sham-treated LNs, perturbation of interstitial flow dramatically decreased expression of VEGF-A in LNs (Fig 5.4A) In addition, perturbation of interstitial flow altered VEGF-A distribution in LNs such that it was markedly confined to the superficial cortex This indicated that during late inflammation, interstitial flow within LNs governed distribution of VEGF-A into the paracortex and medulla To further support this, disruption of interstitial flow was noted to obliterate association of VEGF-A with cortical and medullary sinuses compared to sham-treated LNs (Fig 5.4B) This implies that lymph flow through the

LN could influence the spatial-temporal distribution of VEGF-A during the course of inflammation and, as a consequence, modulate the remodeling of cortical and medullary sinuses

As other groups have described that interstitial flow can modulate FRC organization and function (Tomei et al., 2009) and, in our model, extra-nodal VEGF-A may be transported within FRC fibers and subsequently displayed and/or produced by FRCs ,

we next investigated what might be the repercussions of disrupting interstitial flow on VEGF-A interaction with the fibroblastic reticular network In contrast to sham-treated LNs, perturbation of interstitial flow through LNs ablated VEGF-A co-localization with ER-TR7+ reticular fibers lining the cortical and medullary sinuses (Fig 5.4C) This suggests that LN interstitial flow is an important regulator of both VEGF-A production and presentation by the FRC network

Figure 5.4 Disrupting interstitial flow through DLNS affected VEGF-A localization (A) LV resection resulting in perturbation of interstitial flow, affected

VEGF-A expression and distribution in DLNs Confocal images showed that disruption of interstitial flow in DLNs abolished association of VEGF-A with cortical-medullary sinuses (B) and with FRCs lining them (C) Scale bar in (A) represents 400 µm Scale bar in (B) and (C) represents 50µm L = lumen

Images are representative of 3 independent experiments (n=3)

5.3 Summary

Our data provide, to our knowledge, the first evidence that inflammation when it evolves from early to late phases can induce a differential remodeling of lymphatics, the subcapsular sinuses being expanded first followed by the cortical and medullary sinuses Our data supported a scenario whereby interplay between LN interstitial flow, fibroblastic reticular network and spatial-temporal distribution of VEGF-A formed coordinating forces to drive cortical and medullary sinuses remodeling during late inflammation

During early inflammation, VEGF-A may be transported from its primary site of production, i.e the inflamed peripheral tissue, to the DLNs (Halin et al., 2007) and/or produced by activated B cells or FRCs residing in the cortex of the DLNs (Angeli et al., 2006) At this stage, VEGF-A expression was mainly confined to the subcapsular region of the LN The heparin-binding carboxyl-domain of the predominant VEGF-A isoform, VEGF164 may bound the molecule to the extracellular matrix and limit its access deeper into the LN via the reticular network (Ferrara et al., 2003) In contrast,

at later phases of inflammation, VEGF-A expression was not restricted to the subcapsular region of the LN but extended into the cortical and medullary regions This led us to suspect that differential remodeling of lymphatics may be attributed in part to temporal-spatial changes in VEGF-A distribution in the inflamed LN As further proof, while VEGF-A was noted to co-localize with subcapsular sinuses on both day 4 and 14 after immunization, VEGF-A co-localization with cortical and medullary sinuses was only observed on day 14 post-immunization

We proceeded to show, using surgical experiments, that the temporal-spatial distribution of VEGF-A within the activated LNs during inflammation was dependent

on interstitial flow In addition, interstitial flow may also support the production of VEGF-A by FRCs and/or combined with expansion of the FRC network increase delivery of extra-nodal VEGF-A to the T cell zone Regardless of its source, VEGF-A may be displayed by cortical and medullary sinuses -lining reticular fibers to drive expansion of the sinuses

Chapter 6 Results

Neutrophils can drive lymph node lymphangiogenesis in mice lacking

B cells

Chapter 6 Neutrophils can drive lymph node

lymphangiogenesis in mice lacking B cells 6.1 Introduction

Many immune cells have been described to orchestrate lymphangiogenesis in inflamed peripheral sites and the draining LNs Macrophages, in particular, have been demonstrated to be indispensable in driving lymphangiogenesis in the inflamed periphery (Baluk et al., 2005; Cursiefen et al., 2004b; Cursiefen et al., 2011; Kang et al., 2009; Kataru et al., 2009; Kim et al., 2009; Kubota et al., 2009; Maruyama et al., 2005; Yao et al., 2010) and LNs draining the inflamed sites (Kataru et al., 2009) DCs have been linked to the induction of lymphangiogenesis in various models of tissue inflammation (Baluk et al., 2005; Hamrah et al., 2003; Kanao and Miyachi, 2006; Muniz et al., 2011) T cells have also been reported to induce de novo lymphangiogenesis when recruited into murine thyroids over-expressing CCL21 (Furtado et al., 2007) Although neutrophils have been described to express VEGF-C

in a murine model of chronic airway inflammation (Baluk et al., 2005), a possible involvement in lymphangiogenesis has never been carefully examined Our group (Angeli et al., 2006) and others (Liao and Ruddle, 2006; Shrestha et al., 2010) have shown that B cells are critical for LN lymphangiogenesis occurring early after immunization and sensitization In order to understand if B cells continue to be important mediators of LN lymphangiogenesis during late inflammation, we immunized transgenic mice that are unable to express the µ heavy chain hence lacking

B cells (µMT mice) with CFA/ KLH Unexpectedly, this led to the serendipitous finding that neutrophils play an overlooked but pivotal role in driving lymphangiogenesis

90 days after immunization Notably, this increase in the LEC population in µMT mice was not dependent on LN cellularity but primarily associated with an increased proportion of the LEC population (Fig 6.1, A and B) Analysis of LN sections by immunofluorescence staining for lymphatics was consistent with our flow cytometry data While early expansion of the lymphatic network was clearly greater in inflamed

WT compared to µMT LNs, this gave way to a greater growth of lymphatics in µMT LNs by day 14 post-immunization (Fig 6.1C)

Expansion of the BEC and FRC populations in inflamed µMT LNs occurred in a manner similar to LECs In the early phase of inflammation, expansion of the BEC and FRC populations in µMT LNs was less compared to WT but the converse was seen in the late phases of inflammation (Fig 6.1, D and E)

Figure 6.1 LN remodeling in WT and µMT mice following CFA/ KLH footpad immunization (A) Increase in LN cellularity in WT and µMT mice following

immunization (B) Expansion of LECs population in WT and µMT mice following immunization (C) Immunofluorescence analysis of WT and µMT LN sections (D and E) Expansion of the BEC (D) and FRC (E) populations in inflamed µMT LNs mice following immunization

Results are pooled from 3 independent experiments with 3-4 mice per group in each experiment Student’s t test, *: p<0.05 Error bars represent SD Images are representative of 3 independent experiments (n=3) Scale bar represent 200µm

1.2.2 Lymph node lymphangiogenesis in µMT mice is accompanied by

accumulation of neutrophils and monocytes

We next examined the T cell, DC and myeloid cell populations present in WT and µMT LNs as this may provide clues as to what cells may drive lymphangiogenesis in the absence of B cells We found similar increases in the T cell and DC populations in

WT and µMT LNs at all time points after immunization (data not shown) Coincidentally, we observed a greater increase in the absolute numbers of myeloid cells (defined as CD11bpositive CD11cnegative cells- staining cells) in µMT LNs compared to WT (Fig 6.2A) These myeloid cells expressed intermediate to high levels of Gr-1 (Fig 6.2A) and likely represent neutrophils and/ or monocytes Indeed, when we used Ly6G as a specific marker for neutrophils (Daley et al., 2008; Fleming

et al., 1993), we found that a good proportion of these myeloid cells in µMT LNs were made up of neutrophils (defined as CD11bpositive Ly6Gpositive staining cells) (Fig 6.2B), although monocytes were also present Increase in the absolute numbers of neutrophils in the early days after immunization (days 4 and 7) were initially comparable between WT and µMT LNs (Fig 6.3A) However, by day 14 post-immunization, the increase in neutrophils in µMT LNs (28-fold over control) surpassed that of WT (5-fold over control) and was sustained for 90 days (Fig 6.3A)

A similar scenario was seen when we examined the increase in monocytes (defined as CD11bpositive CD11cnegative Gr-1positive Ly6Gnegative staining cells); increase in monocytes in µMT LNs (18-fold over control) surpassed that of WT (5-fold over control) (Fig 6.3B) and was sustained for 90 days

Figure 6.2 Myeloid cells and neutrophil populations in WT and µMT LNs at baseline and day 14 after immunization as revealed

by flow cytometry (A) Typical dotplots representing the myeloid cells population in WT and µMT LNs at day 14 after immunization

(B) Typical dotplots representing the neutrophil population in WT and µMT LNs at day 14 after immunization Numbers next to gates

indicate percent of cells relative to the total live single cells population

Data shown are representative of 3 independent experiments with 3-4 mice per group in each experiment

Figure 6.3 Myeloid cells and neutrophil populations in WT and µMT LNs after immunization (A) Increase in the neutrophil population in WT and µMT LNs

following immunization (B) Increase in the monocyte population in WT and µMT LNs following immunization

Data shown are pooled from 3 independent experiments with 3-4 mice per group in each experiment Student’s t test, *: p<0.05 Error bars represent SD

Upon staining LN sections for CD11b, we verified that there was a striking infiltration of CD11b+ cells into the µMT compared to WT LNs (Fig 6.4A) More importantly, we noted that these CD11b+ cells interacted very closely with the growing lymphatics in the µMT LNs (Fig 6.4A) Further immunostaining performed

on these same µMT LNs revealed that the CD11b+ cells which interacted closely with lymphatics, were also Ly6G (Fig 6.4B) and Gr-1 (Fig 6.4C) expressing This suggested that the CD11b+ cells that accumulated in µMT LNs at day 14 post-immunization consisted mainly of neutrophils and monocytes In addition, their close proximity to the growing lymphatics suggested that these cells may functionally drive lymphangiogenesis in the absence of B cells

Figure 6.4 Myeloid cells and neutrophil populations in WT and µMT LNs at day 4 and 14 after immunization (A) Increased

infiltration of CD11b+ cells in µMT compared to WT LNs on day 14 post-immunization The CD11b+ cells in µMT LNs were in close interaction with lymphatics (B and C) Immunostaining performed on LN sections in (A) revealed that the CD11b+ cells present in µMT LNs were also Ly6G+ (B) and Gr-1+ (C) expressing Data shown are representative of 3 independent experiments (n=3) Scale bars represent 100µm for (A) and 200µm for (B) and (C)

6.2.3 Neutrophils are critical for lymph node lymphangiogenesis in the absence

of B cells

As expansion of neutrophils in µMT LNs at day 14 was the most dramatic amongst the immune cells and this coincided with a surge in lymphangiogenesis, we hypothesized that neutrophils may compensate for B cells to drive lymphangiogenesis during late inflammation In order to determine if neutrophils are truly important for lymphangiogenesis in a model where B cells are absent, we treated µMT mice with the neutrophil-depleting NIMP-R14 MAb or a control rat IgG for 2 weeks after immunization (Fig 6.5A) Blood profiles for both NIMP-R14 MAb and control rat IgG-treated mice were performed on days 0, 7 and 14 to assess depletion of neutrophils Treatment with NIMP-R14 MAb effectively depleted neutrophils in the peripheral blood for the 2 weeks after immunization (Fig 6.5B) and attenuated accumulation of neutrophils in µMT LNs at day 14 (Fig 6.5D) Compared to the 16-fold increase in LEC numbers in control rat IgG treated µMT mice at day 14, treatment with NIMP-R14 MAb abrogated increase in LEC numbers to only 2.6-fold (Fig 6.5C) Treatment of µMT mice with NIMP-R14 MAb also affected expansion of the BEC and FRC populations compared to control rat IgG-treated mice It is noteworthy that attenuating neutrophil accumulation led to a paradoxical increase in the population of non-granulocytic myeloid cells (defined as defined as CD11bpositiveCD11cnegative Ly6Gnegative and either GR-1 intermediate or F4/80 positive staining cells by flow cytometry) within µMT LNs These non-granulocytic myeloid cells likely consisting of both monocytes and macrophages, escalated from a 10-fold increase in control rat IgG-treated mice to a 40-fold increase in NIMP-R14-treated mice (Fig 6.5D) However, in spite of this increased accumulation of non-granulocytic myeloid

cells in the µMT LNs, they were not able to compensate for neutrophils in driving lymphangiogenesis

Figure 6.5 Neutrophils are critical for LN lymphangiogenesis in the absence of B cells (A) Diagram depicts treatment of µMT mice with NIMP-R14 MAb or control

rat IgG after immunization (B) Typical dot plot representing the neutrophil population in the peripheral blood of immunized µMT mice treated with NIMP-R14 MAb or control rat IgG Numbers next to gates indicate percent of cells relative to the total live single cells population (C) Expansion of the LEC, BEC and FRC populations in µMT LNs were all abrogated by treatment with NIMP-R14 MAb (D) Neutrophil and monocyte populations in LNs of µMT mice treated with NIMP-R14 MAb or control rat IgG

Data shown are pooled from 2 independent experiments with 5 mice per group in each experiment Student’s t test, *: p<0.05 Error bars represent SD

6.2.4 Increased accumulation of neutrophils within µMT lymph nodes is

mediated by increased expression of neutrophil chemoattractants

We wanted to study which chemokines may mediate the accumulation of neutrophils

in µMT LNs We therefore determined the expression of known neutrophil chemoattractants, the ELR+ chemokines, namely CXCL1, CXCL2 and CXCL5 Compared to control, WT whole LNs demonstrated little mRNA up-regulation of CXCL1 (Fig 6.6A), CXCL2 (Fig 6.6B) and CXCL5 (Fig 6.6C) during inflammation, consistent with the observation that there was little infiltration of neutrophils into WT LNs after immunization In contrast, analysis of µMT whole LNs revealed mRNA up-regulation of CXCL1 (Fig 6.6A), CXCL2 (Fig 6.6B) and CXCL5 (Fig 6.6C) which peaked at day 10 post-immunization and was sustained until day 60 This indicated that recruitment of neutrophils into µMT LNs by day 14 till later days of immunization was probably triggered by upregulation of these ELR+ chemokines

As T cells made up the largest population of cells in the µMT LNs, we next asked if upregulation of ELR+ chemokines was related to polarization of the T cells to a particular subset(s) We found similar expression of Th1, Th2, Treg and Th17 specific transcriptional factors, namely T-bet, Gata-3, FOXP3 and RORγ in WT and µMT LNs

at day 10 immunization (Fig 6.7) This suggests that at day 10 immunization, there was comparable representation of the different T cell subsets in

post-WT and µMT LNs and this was therefore not a factor governing migration of neutrophils into µMT LNs

Figure 6.6 mRNA expression of various neutrophil chemoattractants in WT and µMT LNs RT-PCR analysis revealed a significant increase in the mRNA levels of

ELR chemokines, CXCL1 (A), CXCL2 (B) and CXCL5 (C) in µMT LNs by day 10 post-immunization compared to control Differences between immunized µMT LNs compared to control were analysed by student’s t test and is represented by *: p< 0.05,

**: p< 0.01.Data is representative of 2 independent experiments consisting of 3 mice each experiment Error bars represent SD

Figure 6.7 mRNA expression of Th1 (T-bet), Th2 (Gata-3), Treg (FOXP3) and Th17 (RORγ) specific transcriptional factors in WT and µMT LNs RT-PCR

analysis revealed similar mRNA levels of these transcriptional factors in µMT and

WT LNs at day 10 post-immunization

Data is representative of 2 independent experiments consisting of 3 mice each experiment Error bars represent SD

6.2.5 Neutrophils and non-granulocytic myeloid cells cooperate to drive

lymphangiogenesis in the absence of B cells

Other than neutrophils, we also saw a prominent expansion of monocytes in µMT LNs This, coupled with the wealth of data to demonstrate that macrophages/ monocytes play indispensable roles in lymphangiogenesis prompted us to explore if monocytes may cooperate with neutrophils to drive lymphangiogenesis in the absence

of B cells To this end, we treated µMT mice with the monocyte and depleting AFS98 MAb or a control rat IgG for 2 weeks after immunization (Fig 6.8A) Because the monoclonal antibody binds to CSF-R and blocks its signaling, it will induce depletion of both monocytes and macrophages (Kubota et al., 2009; Sudo

macrophage-et al., 1995) Blood profiles for mice were performed on days 0, 7 and 14 to assess depletion of blood monocytes Treatment with AFS98 MAb effectively depleted circulating monocytes (identified as CD11bpositive F4/80positive CD115positive) during treatment (Fig 6.8B) and attenuated accumulation of non-granulocytic myeloid cells

in µMT LNs at day 14 (Fig 6.8D) Compared to control rat IgG treated µMT mice, treatment with AFS98 Mab attenuated increases in the LEC population (Fig 6.8C) Treatment of µMT mice with AFS98 MAb also diminished expansion of the BEC and FRC populations compared to control rat IgG-treated mice Attenuating accumulation

of non-granulocytic myeloid cells did not alter neutrophil recruitment to the µMT LNs (Fig 6.8D) While neutrophils continued to be recruited to the µMT LNs in the AFS98-treated mice, they were not able to compensate for non-granulocytic myeloid cells to drive lymphangiogenesis

Figure 6.8 Non-granulocytic myeloid cells are critical for LN lymphangiogenesis

in the absence of B cells (A) Diagram depicts treatment of µMT mice with AFS98

MAb or control rat IgG after immunization (B) Typical dot plot representing the monocyte population in the peripheral blood of immunized µMT mice treated with AFS98 MAb or control rat IgG Numbers next to gates indicate percent of cells in relative to the total live single cells population (C) Expansion of the LEC, BEC and FRC populations in µMT LNs were all abrogated by treatment with AFS98 MAb (D) Non-granulocytic myeloid cells and neutrophil populations in LNs of µMT mice treated with AFS98 MAb or control rat IgG

Data shown are pooled from 2 independent experiments with 3-4 mice per group in each experiment Student’s t test, *: p< 0.05 Error bars represent SD

in the absence of B cells, play an indispensable role in driving LN lymphangiogenesis

Although neutrophils were indispensable for the occurrence of lymphangiogenesis in µMT LNs, it is unlikely that they were the only cells driving the process Depletion of monocytes and macrophages by the use of AFS98 MAb inhibited lymphangiogenesis

in the µMT LNs, indicating that these cells play an equally critical role What is more likely is that both neutrophils and monocytes/ macrophages act in concert to drive inflammatory lymphangiogenesis in the µMT LNs and in the absence of either, lymphangiogenesis is effectively crippled

As lymph nodes do not form the de facto homing lymphoid organs for neutrophils, we were interested in what chemokine signals may mediate neutrophil migration into LNs We found that B cell deficiency during inflammation was associated with an increased expression of ELR+ chemokines which are known potent neutrophil chemoattractants That we did not find a skewing of the T cells towards any particular subset(s) in µMT compared to WT LNs, favours the notion that B cells exerts direct suppressive effects on neutrophil trafficking through inhibition of ELR+ chemokines production

Chapter 7 Results

Neutrophils drive lymphangiogenesis in the inflamed

periphery of wild type mice

Chapter 7 Neutrophils drive lymphangiogenesis in the

inflamed periphery of wild type mice

7.1 Introduction

Having established that neutrophils play a non-redundant role in driving LN lymphangiogenesis in the absence of B cells, we wanted to study if neutrophils play a similar role in driving lymphangiogenesis in normal inflammatory conditions Because neutrophils do not traffic to lymph nodes under normal inflammatory conditions (Fig 6.2 and 6.3), we suspect that they may not be the key immune cells driving LN lymphangiogenesis Therefore we also chose to examine for the occurrence of lymphangiogenesis in peripheral sites where an abundance of neutrophils could be found during inflammation

B cell populations (Fig 7.1F)

We hypothesized that during inflammation in the absence of immune deficits, neutrophils may play a pivotal role in organizing lymphangiogenesis in sites where they could be abundantly found We noted that immunization with CFA/ KLH elicited

a recruitment of neutrophils into the inflamed footpad, which appeared to peak between day 7 and 10 after immunization (Fig 7.2) Lymphangiogenesis in the inflamed footpad seemed to lag behind neutrophils accumulation and appeared to peak at day 10 to 14 after immunization (Fig 7.2) Close interactions between neutrophils and the growing lymphatic vessels were also noted In all, this implies that neutrophils may have an important role in mediating lymphangiogenesis in the inflamed peripheral site

Figure 7.1 Neutrophils are not critical for LN lymphangiogenesis in normal inflammatory conditions (A) Diagram depicts treatment of WT mice with NIMP-

R14 MAb or control rat IgG after immunization (B) Typical dot plot representing the neutrophil population in the peripheral blood of immunized WT mice treated with NIMP-R14 MAb or control rat IgG Numbers next to gates indicate percent of cells relative to the total live single cells population (C) Expansion of the LEC, BEC and FRC populations in WT LNs following NIMP-R14 MAb or control rat IgG treatment (D) WT LNs of mice treated with NIMP-R14 MAb was more swollen compared to those treated with control rat IgG Scale bar represent 2mm (E and F) LN cellularity (E) and T and B cells populations (F) in mice treated with NIMP-R14 MAb or control rat IgG

Data shown are pooled from 2 independent experiments with 3-4 mice per group in each experiment Student’s t test, *: p< 0.05 Error bars represent SD

Figure 7.2 Neutrophils accumulate in the inflamed footpads following CFA/ KLH immunization Immunization with CFA/ KLH elicited a recruitment of

neutrophils into the inflamed footpad, which appeared to peak between day 7 and 10 Lymphangiogenesis in the inflamed footpad seemed to lag behind accumulation of neutrophils, peaking at day 10 to 14 after immunization Data shown are

representative of 3 independent experiments (n=3) Scale bars represent 100µm