SEARCH

SEARCH BY CITATION

Keywords:

  • Monocytes;
  • NOD;
  • Migration;
  • Chemokines;
  • IL-10

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The accumulation of macrophages (MΦ) and dendritic cells (DC) in the pancreas plays a crucial role in the pathogenesis of autoimmune diabetes. We studied the recruitment of monocytes, MΦ and DC to sites of inflammation, i.e. the peritoneal cavity and a subcutaneously elicited air pouch in the NOD mouse model of autoimmune diabetes. The leukocyte recruitment was studied from 1 to 7 days after injection of thioglycollate (peritoneum), C5a (peritoneum, air pouch), CCL2 and CCL3 (air pouch). C57BL/6 and BALB/c mice served as controls. Morphological and flow cytometric analysis of the recruited cells was performed, IL-1β, TNF-α, IL-6, IL-12 and IL-10 in exudates measured, and in vitro CCL2-chemotaxis of exudate MΦ (Boyden chamber) determined. NOD mice were strongly impaired in the recruitment of MΦ, DC, monocytes, and granulocytes. Chemokine-injected air pouches of NOD mice showed an increased IL-10 and a decreased IL-1β level, while the other cytokines were normally or very lowly expressed. In addition, NOD exudate MΦ displayed an impaired in vitro CCL2-induced migration. Our data show that NOD mice have an impaired ability to recruit leukocytes into sites of inflammation elicited in the peritoneum and the air pouch. A raised IL-10/IL-1β ratio at these sites and a deficient migratory capacity of NOD monocytes are important determinants in this impairment.

Abbreviations:
TG:

Thioglycollate

SSC:

Side scatter

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Macrophages (MΦ) and dendritic cells (DC) play a crucial role in the pathogenesis of type-1 diabetes. In rodent models, MΦ and DC are the first cells to accumulate in the pancreas and are predominantly situated at the edges of the islets 1, 2. The cells play a role in the initiation and perpetuation of the islet autoimmune response by taking up islet antigens and presenting these antigens to naive islet-specific T cells in the draining lymph nodes 3. In later phases of the disease DC and MΦ form an integral part of the lymphocytic insulitis. In these phases, the MΦ assist the lymphocytes in the destruction of the β cells. It is assumed that the islet-associated MΦ and DC are mainly derived from monocytes recruited from the blood stream.

The recruitment of monocytes into sites of inflammation involves a complex series of steps. After margination from the circulation, the cells start to roll on the endothelium and firmly adhere to it 4. Thereafter, the cells squeeze through the endothelial layer and diapedese into the tissues. Selectins, integrins and chemokines play prominent roles in these processes 46. The early infiltrating monocytes induce endothelial cells to secrete chemokines and to up-regulate the expression of vascular adhesion molecules by the production of inflammatory compounds, such as interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and myeloid cell-related proteins (MRP) 710. This facilitates a further infiltration of monocytes. The recruited monocytes differentiate into inflammatory MΦ or DC that produce cytokines like IL-12 and TNF-α to maintain the inflammatory environment and to initiate an immune response 11, 12. Anti-inflammatory cytokines produced in the inflammation, such as IL-10 and transforming growth factor (TGF)-β, may exert suppressive effects by a down-regulation of the production of pro-inflammatory cytokines and thus limit the inflammatory reaction 13, 14.

The non-obese diabetic (NOD) mouse is a widely used animal model to study type-1 diabetes. Here we report a study on the recruitment of inflammatory cells, including monocytes, into two separate sites of inflammation in the NOD mouse, i.e. into the peritoneal cavity 15 and into an air pouch elicited subcutaneously 16, 17. Inflammatory cells were attracted to these sites by injecting thioglycollate (TG) (peritoneum), the potent chemoattractant C5a (peritoneum, air pouch) or the chemokines CCL2 or CCL3 (MIP-1α) (air pouch). Interestingly, NOD mice were in all instances strongly impaired in the recruitment of monocytes (as well as that of granulocytes) into these sites of inflammation. IL-10 was clearly raised in the air pouch of NOD mice, while IL-1β levels were decreased. Anti-IL-10 treatment only partially restored the severely hampered monocyte recruitment, indicating that the role of this cytokine is limited in the recruitment disturbances.

Mouse blood monocytes can be subdivided into two populations, based on the differential expression of the Ly-6C molecule 18; the immature monocytes (Ly-6Chi) and the mature monocytes (Ly-6Clow). Immature monocytes preferentially migrate to sites of inflammation 18, 19. Here we also report on the effects of the induction of the inflammatory reaction in the air pouch on the frequency of Ly-6Chi cells in the circulation of NOD mice. The outcomes urged us to study the in vitro (Boyden-chamber) chemokine-induced migratory capability of NOD exudate MΦ.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Impaired influx of inflammatory cells into the peritoneal cavity in NOD mice

To study the in vivo recruitment of inflammatory cells, a sterile inflammation was induced by injecting TG intraperitoneally (i.p.). In control C57BL/6 mice, inflammatory cells, including granulocytes and monocytes, were recruited early and in large numbers into the peritoneal cavity after TG injection, as determined by morphological analysis using differential cell count (Fig. 1B, C; see insets for the morphological identification). The recruitment of monocytes and granulocytes was followed by an increase in the numbers of MΦ with a maximum at 4 days (Fig. 1D).

thumbnail image

Figure 1. NOD mice (5–7 weeks old) show a strongly decreased influx of inflammatory cells after TG injection (A). Combined result of two experiments is shown (n=5/group, mean ± SEM). Morphological analysis (see insets for representative example) shows a decreased influx of granulocytes (B), monocytes (C) and MΦ (D) in NOD mice. Data shown are representative of one experiment with three mice per group (mean ± SD). *p<0.05 as determined with Student's t-test.

Download figure to PowerPoint

NOD mice showed a strongly decreased recruitment of inflammatory cells after TG injection, as shown in Fig. 1A–D. Since the autoimmune-related cellular infiltrates in the pancreas, salivary glands and other autoimmune inflammations of the NOD might be responsible for the reduced recruitment into the peritoneum after TG administration (via e.g. redistribution of the cells), we also injected TG into very young mice (3 weeks) prior to the development of such cellular autoimmune infiltrates. These young NOD mice showed a decreased recruitment of inflammatory cells into the peritoneum after TG injection (data not shown). The low influx of cells could not be explained by a delayed inflammatory response of NOD mice, since the kinetics of inflammation onset were similar in NOD and C57BL mice (Fig. 1A).

With regard to the various subsets of inflammatory cells, the influx of granulocytes in the peritoneal cavity of NOD mice was strongly decreased compared to that of the C57BL/6 mice (Fig. 1B), while the number of monocytes did not increase at all (Fig. 1C). There was a small increase in the number of MΦ at day 1, but much reduced compared to C57BL/6 (Fig. 1D). Upon injection of another inflammatory substance, i.e. the chemoattractant C5a into the peritoneum, NOD mice also displayed a strongly reduced recruitment of granulocytes, monocytes and MΦ in the peritoneal cavity in comparison to the control C57BL/6 mice (data not shown).

Severely reduced recruitment of inflammatory cells in the air pouch in NOD mice

To investigate whether reduced recruitment of inflammatory cells was a peritoneum-specific or generalized phenomenon in NOD mice, we also studied another controlled environment for inducing an inflammation, by creating a pouch filled with sterile air on the back of the mouse 16, 17. The chemoattractant C5a was injected into the air pouch, which after 8 h in control C57BL/6 mice resulted in a threefold higher influx of inflammatory cells compared to animals that were injected with the vehicle only (Fig. 2A). Again granulocytes were the early infiltrating cells, while monocytes and MΦ were the predominant cells after 48 h as determined by morphological analysis using differential cell count (Fig. 2B–D). In NOD mice, a lower number of granulocytes and monocytes were recruited in the air pouch after C5a injection (Fig. 2B, C); the number of MΦ was only slightly and not significantly reduced (Fig. 2D).

thumbnail image

Figure 2. NOD mice (5–7 weeks old) show a strongly impaired recruitment of inflammatory cells into the air pouch 24 h after C5a injection (A). Morphological analyses shows less granulocytes (B) and monocytes (C) in NOD compared to C57BL/6. MΦ (D) increase similarly in time in C57BL/6 and NOD mice. Data are represented as mean ± SD (n=2/time point). Dashed line represents C57BL/6, solid line represents NOD, *p<0.05 relative to NOD as determined with Student's t-test.

Download figure to PowerPoint

We investigated this air pouch model further, using another potent granulocyte and monocyte chemoattractant, CCL3. This experiment was carried out with an additional control strain, i.e. the BALB/c mouse, to exclude the possibility that the effect was specific for the C57BL/6 mouse. Again NOD mice showed a strongly reduced influx of inflammatory cells in the air pouch by 24 h after injection in comparison to these two control strains (Fig. 3A). This reduced recruitment again involved both the granulocytes and monocytes (Fig. 3B, C, Table 1).

thumbnail image

Figure 3. The accumulation of cells 24 h after CCL3 injection is hampered in NOD (5–7-week-old) mice (A). Granulocytes (B) and monocytes (C) are not attracted in NOD mice. In response to CCL2, NOD mice show a deficient accumulation of cells into the air pouch after 24 h (D). The accumulation of monocytes to CCL2 is almost absent in NOD (E). Data are cumulative of two individual experiments and represented as mean ± SEM (n=6/group in A and C; n=4/group in B, C, E). *p<0.05 relative to NOD as determined with Mann-Whitney (A, D) or Student's t-test (B, C, E).

Download figure to PowerPoint

Table 1. Recruitment of cells in absolute numbers in response to CCL3
Total cells (×10–6 cells)a)Granulocytes (×10–6 cells)Monocytes (×10–6 cells)
  1. a)Data are shown as averages ± SEM and each group represents at least six animals (5–7 weeks of age).b)p<0.05 (Mann-Whitney) relative to NOD CCL3-injected animals.c)p<0.05 (Mann-Whitney) relative to NOD control (vehicle injected) animal.

ControlCCL3ControlCCL3ControlCCL3
NOD/LTj1.56±0.341.53±0.420.92±0.140.84±0.280.27±0.050.25±0.08
C57BL/62.21±0.564.42±0.93b)0.59±0.011.93±0.40b)0.06±0.01c)0.16±0.03
BALB/c3.55±0.51c)9.89±1.13b)1.87±0.543.69±0.66b)0.12±0.050.39±0.09

Finally, CCL2, a specific monocyte chemoattractant, was tested in the air pouch model. Again NOD mice showed a strongly diminished influx of cells into the air pouch after 24 h in comparison to C57BL/6 and BALB/c mice, and this reduction mainly involved the influx of monocytes, which was very low in NOD after CCL2 injection (Fig. 3D and E, respectively). No significant recruitment of granulocytes was detected in response to CCL2 injection in either control mice or NOD mice (data not shown).

Inflammation in the air pouch lowers the number of inflammatory circulating monocytes in control, but not in NOD, mice

The monocytes that were recruited into the peritoneal cavity and into the air pouch derive from monocytes that circulate in the blood. These cells are direct precursors of MΦ and DC 18. Therefore, we studied whether the lower cell numbers found at the inflammatory site in the NOD mouse originated in lower recruitment of the cells from the circulation. Monocytes were defined in flow cytometry as SSClowCD11bhiLy-6G cells and were divided into two populations based on the expression of Ly-6C 18. Of these, the Ly-6Chi monocytes represent the immature monocytes that have recently emigrated from the bone marrow that are predominantly recruited into sites of inflammation 18. Fig. 4 presents the gating method that we used to analyze the various types of leukocytes in the circulation or accumulated in the air pouch.

thumbnail image

Figure 4. Flow cytometry of different cell types in air pouch exudates. Based on SSC and Ly-6G profile, three populations are defined (R1, R2 and R3). Monocytes are defined as CD11bhiLy-6G-F4/80low/medCD11clow in the R2 gate and based on their Ly-6C expression, two subpopulations are identified. Granulocytes are defined in the R1 gate and by exclusion from gate R3 on their high SSC profile. MΦ and DC are defined using the gate R3 after exclusion of the granulocytes in gate R4. MΦ are F4/80hiCD11clow (R5) and DC F4/80medCD11chi (R6). Representative figures are shown of a BALB/c mouse 24 h after CCL3 injection.

Download figure to PowerPoint

The monocytes that were present in the air pouch either before or after the injection of CCL3 were mainly of the Ly-6Chi phenotype in the three mouse strains studied (Fig. 5A), thus demonstrating that, also in the NOD mouse, the Ly-6Chi monocytes preferentially accumulate at the inflammation site. In the blood of control mice a reduction of about 18% of the Ly-6Chi monocytes was observed 24 h after CCL3 injection into the air pouch (Fig. 5B). NOD mice, however, only showed a mild reduction of 7% of the circulating Ly-6Chi monocytes (Fig. 5B), which is consistent with an impaired recruitment of such monocytes into the air pouch. Interestingly, the frequency of Ly-6Chi monocytes was decreased in the blood of NOD mice in comparison to control mice (Fig. 5B). However, when absolute numbers of these cells were calculated, the blood of NOD mice contained significantly less Ly-6Clow monocytes than C57BL/6 or BALB/c (not significantly) control mice (data not shown).

thumbnail image

Figure 5. The monocytes that are present in the air pouch either after CCL3 injection or after injection of vehicle are Ly-6Chi (A). The Ly-6Chi monocytes are present at lower frequency in the blood in NOD mice and, after CCL3 injection, a reduction in their frequency is observed, which in control mice is more pronounced (B). Representative histograms are shown (controls n=3; CCL3 n=6).

Download figure to PowerPoint

Low numbers of DC and MΦ in the air pouch of NOD mice

To exclude the possibility that the decreased number of monocytes in inflamed air pouches in NOD mice could be the result of an enhanced differentiation of these cells into inflammatory MΦ or DC, we performed a more detailed analysis of the cells in the CCL2-injected air pouches, using flow cytometry. DC were defined as SSCmedCD11bmed/hiF4/80medCD11chi cells and MΦ as SSCmedCD11bhiF4/80hiCD11clow cells (Fig. 4). When CCL2 was injected in control C57BL/6 and BALB/c mice an increase in the number of both MΦ and DC was observed after 24 h (Fig. 6A, B). These increases were absent in NOD mice (Fig. 6A, B).

thumbnail image

Figure 6. The air pouch of C57BL/6 and BALB/c contains a high number of MΦ (A) and DC (B) after CCL2 injection, while those of NOD mice do not. MΦ and DC are defined as shown in Fig. 4. Data are cumulative of two experiments and are represented as mean ± SEM; each bar represents at least four animals (5–7 weeks old), *p<0.05 as determined with Student's t-test.

Download figure to PowerPoint

A partial role for IL-10 in the reduced inflammatory cell recruitment in NOD air pouches

The production of cytokines at a site of inflammation plays an important role in the attraction of inflammatory cells into that site. Therefore, we investigated the production of cytokines in the air pouch by performing ELISA on the exudate fluids retrieved from the air pouches. Fig. 7A shows that the NOD mouse failed to produce IL-1β in the air pouch after CCL2 injection, in contrast to C57BL/6 and BALB/c mice. NOD mice exhibited a significantly higher production of IL-10 in the air pouch in comparison to the control mice (Fig. 7B). To investigate the primary source of the IL-10 in the air pouch, we performed intracellular flow cytometry staining of the cells isolated from the exudates of CCL2-injected NOD mice. Some of the F4/80low/med cells contained intracellular IL-10 and additional analyses demonstrated that these cells were CD11cneg/Ly-6Chi monocytes (Fig. 7C and data not shown). The TNF-α production was not affected in the air pouches of the NOD mice (Fig. 7D). In all mouse strains, the levels of IL-6 and IL-12 in the air pouch exudates were too low to be reliably measured (data not shown). Upon injection of CCL3 into the air pouches, results similar to those obtained with CCL2 were found for IL-1β, IL-10 and TNF-α (data not shown).

thumbnail image

Figure 7. The air pouch of control mice at 24 h after CCL2 injection shows an increased production of IL-1β; however, this is not seen in NOD (A). NOD mice produce higher amounts of IL-10 in comparison to C57BL/6 and BALB/c mice (B). A representative flow cytometric analysis of intracellular staining for IL-10 (gated for CD11b+ cells) shows that monocytes (CD11bhiF4/80medCD11clow) are producing IL-10, while MΦ (CD11b+F4/80hiCD11clow) do not (C). Figures shown are representative of five NOD mice 24 h after injection with CCL2. The production of TNF-α in the air pouch of NOD mice is similar to control mice (D). Data are represented as mean ± SEM (n=5/group; 5–7 weeks old). *p<0.05 relative to NOD as determined with Student's t-test.

Download figure to PowerPoint

Since IL-10 affects the production of IL-1β 13, we argued that the high concentration of IL-10 in the air pouch might be responsible for the decreased IL-1β production and the diminished influx of inflammatory cells in the air pouch of NOD mice. To approach this experimentally, we injected recombinant mouse IL-10 together with CCL2 in the air pouch of control mice. Indeed, we observed a decrease in the recruitment of inflammatory cells (Fig. 8A), but this did not seem to work via a reduction in IL-1β expression (Fig. 8B). In contrast, neutralization of IL-10, by injection of a neutralizing antibody to IL-10 together with CCL2, caused only a slight increase in the number of inflammatory cells (Fig. 8A), but a significant increase the IL-1β levels (Fig. 8B) in the air pouch of NOD mice.

thumbnail image

Figure 8. Neutralization of IL-10 does not restore the cell recruitment towards CCL2 to the level of C57BL/6 mice (A). Addition of recombinant IL-10 to the air pouch of C57BL/6 mice decreases the recruitment towards the control level (A). Blocking IL-10 raises the level of IL-1β in the air pouch of NOD to a similar level of C57BL/6 mice (B). Addition of recombinant IL-10 in the air pouch of C57BL/6 does not lower the amount of IL-1β (B). Mean ± SD (n=6) are shown. Data in (B) are presented relative to the amount of IL-1β that is observed in control-injected animals. Statistical significance was determined with Kruskall-Wallis test and differences between groups using the Mann-Whitney test.

Download figure to PowerPoint

Taken together, the manipulation of IL-10 in the control C57BL/6 mice influenced the influx of inflammatory cells, but not through IL-1β. In the NOD mouse, a blockade of IL-10 contributed to the restoration of the IL-1β level, but this was not sufficient to achieve a recruitment of inflammatory cells into the air pouch compared to control C57BL/6.

NOD exudate MΦ show an impaired in vitro CCL-2 induced migratory capability

The impaired recruitment of inflammatory cells in NOD might be due not only to specific alterations in the air pouch environment (such as the cytokine environment), but also to intrinsic migratory aberrancies of the inflammatory cells. Therefore, we performed in vitro chemotaxis using a Boyden chamber. Since these experiments require more blood monocytes than can be obtained practically, we used recently emigrated monocytes, i.e. peritoneal exudate MΦ that had been elicited with TG. We observed that NOD MΦ showed a decreased migration in vitro towards CCL2 in comparison to C57BL/6 mice (Fig. 9).

thumbnail image

Figure 9. Peritoneal exudate MΦ of NOD mice show a decreased migration towards CCL2 compared to C57BL/6 control mice using a Boyden chamber chemotaxis assay. Data are represent mean ± SD (n=3), *p<0.05 relative to NOD as determined with Student's t-test.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This study shows that NOD mice exhibit a strongly reduced recruitment of monocytes, but also of granulocytes, into the peritoneal cavity and into artificially made skin air pouches after injection of various chemokines (C5a, CCL3 and CCL2), and after injection of the nonspecific inflammatory compound TG. Our air pouch model confirmed that the recruited monocytes are of the inflammatory Ly-6Chi subset that had recently emigrated from the bone marrow. During such recruitment, the number of Ly-6Chi monocytes in the circulation decreases concomitantly, shifting the balance from Ly-6Chi in the direction of Ly-6Clow cells. This inflammation-induced shift in blood monocyte subsets was rarely observed in NOD mice after eliciting inflammation. This observation additionally supports the view that the NOD mouse is severely hampered in the recruitment of monocytes to these sites of inflammation. NOD mice may have a reduced potential to recruit monocytes due to the accumulation of monocytes and monocyte-derived DC and MΦ in the autoimmune insulitis, sialoadenitis or other autoimmune inflammations characteristic of the NOD mouse. However, our experiments show that 3-week-old mice, which at this age do not yet show such autoimmune inflammation, also display an inability to recruit monocytes into the peritoneal cavity. Moreover, granulocytes were also not recruited, and granulocytes are rarely seen in NOD autoimmune inflammations.

In relation to the impaired recruitment, there was no increased production of IL-1β in the artificially induced air pouch in NOD mice. This low level of IL-1β likely contributes to the decreased recruitment of inflammatory cells, since IL-1β induces the expression of adhesion molecules on endothelial cells and thus plays a role in the influx of leukocytes from the blood 8, 9. We also observed an increase in IL-10 production in NOD mice. This cytokine has been implicated in a reduced trafficking of leukocytes 20, 21. The increased IL-10 production observed in the CCL2-inflamed air pouch of NOD mice may even be considered a primary abnormality since IL-10 is able to suppress the local production of pro-inflammatory cytokines, such as that of IL-1β 13. Indeed, neutralizing IL-10 with antibodies at the site of inflammation increased the level of IL-1β in the air pouch in NOD mice; however, the influx of cells did not reach the level seen in control mice. Our findings therefore indicate a definite, but only limited, role of the enhanced local IL-10 production in the reduced influx of inflammatory cells in NOD mice, and imply that other factors must also be involved. The cells responsible for the altered IL-10 and IL-1β production in the NOD air pouch have not yet been studied, but, although few in number, the MΦ and monocytes present in the air pouch are likely candidates, since MΦ and monocytes are in general prime sources of these cytokines.

What factors other than cytokines play a role in the severely hampered recruitment of inflammatory cells, including that of monocytes, MΦ and DC, in the artificially induced inflammations in the NOD mouse? NOD mice exhibit a C5 deficiency, a constitutive lower number of circulating inflammatory monocytes, a defective differentiation of DC and MΦ, an altered expression of adhesion molecules on endothelium in inflammation and a diminished chemotactic responsiveness of MΦ and DC, and these are all factors that could be responsible.

NOD mice have a complete C5 deficiency 22, which could play a role in the hampered recruitment response to C5a described here. However, the accumulation of inflammatory cells after injection of other chemoattractants, such as CCL2 and CCL3 was also defective.

In the steady-state, non-inflammatory condition, a constitutive decreased number of inflammatory monocytes is observed in the blood of NOD mice compared to that of control mouse strains (this report), which might certainly be a factor of importance for the reduced recruitment of the Ly-6Chi monocytes into the inflammations. However, the lower quantity of Ly-6Chi blood monocytes is not the decisive factor, since, of those that are present, a low percentage did leave the circulation and entered the air pouch inflammation. Thus, the NOD mice are able to recruit Ly-6Chi monocytes into inflammation, but in strongly decreased numbers, suggesting that not so much the number of circulating Ly-6Chi monocytes, but rather the migratory potential of the monocytes is of importance in the decreased monocyte recruitment.

Mouse monocytes have the potential to differentiate rapidly into DC 23, raising the possibility that the decreased monocyte recruitment in comparison to control mice could be due to an increased differentiation of monocytes into descendent DC. However, we did not find increases in the number of DC or MΦ in the air pouch after CCL2 injection in NOD mice. Moreover, there is ample evidence that the development of precursors to descendent DC and MΦ is hampered in the NOD mouse 2426.

A poor emigration of the cells from the blood stream could also be due to aberrancies of the NOD endothelial cells, such as a low expression of adhesion molecules. However, there is ample evidence in the literature showing that adhesion molecules are over-expressed on endothelial cells of the NOD mouse during inflammations, even though these studies concentrated on the infiltrated islets 27, the thyroiditis 28 or the sialoadenitis 27. We have not determined the expression levels of adhesion molecules in the artificially induced inflammations used here.

Last but not least, aberrancies in the inflammatory cells themselves may have played a role in the decreased recruitment of cells in the artificially induced inflammations. A lower responsiveness to the chemokines used or an altered capability to adhere to endothelial cells may have been causes for "lazy leukocytes" in the NOD mouse. We recently found a lower chemotactic responsiveness of bone marrow-derived DC towards the chemokines CCL2 and CCL19 (manuscript in preparation), and here we show that also NOD exudate MΦ display a reduced migratory response to CCL2 in vitro. A diminished chemotactic response of monocytes from type 1 diabetes patients to casein and C5a in a Boyden chamber 29, and to zymosan-activated medium using an underagarose assay 30 have been reported previously. In addition, in type 1 diabetes patients, we found a decreased responsiveness of monocytes to fMLP and CCL2, but not to CCL3, CCL4 and CXCL12 using the various migration assays (manuscript in preparation). Also a diminished fMLP-induced cytoskeletal rearrangement of monocytes has been observed in autoimmune thyroiditis patients 31, 32 and in type 1 diabetes (unpublished observations). With regard to a putative altered adhesiveness of inflammatory cells to endothelium, we have actually found a raised adhesiveness of monocytes of type 1 diabetes patients to endothelium and the extracellular matrix protein fibronectin (unpublished observations and 33).

It is also important to note the contrast that exists between the poor recruitment of NOD monocytes, MΦ and DC into artificially induced acute inflammations reported here, and the well-established "spontaneous" accumulation of monocytes, MΦ and DC in the pancreas and other sites of chronic autoimmune inflammation of the NOD mouse. It is generally assumed that the MΦ and DC in these chronically inflamed tissues of NOD mice are derived from infiltrating monocytes and/or precursors circulating in the blood. However, it cannot be ruled out that at least part of the accumulation of DC and MΦ stems from the proliferation of local precursors. Such local precursors have been found in the liver 34, the thymus 35 and the peritoneal cavity 36.

There are also other not-mutually exclusive possibilities to explain the difference. An accumulation of monocytes, MΦ and DC at a site of inflammation is not only the outcome of an influx of precursors from the blood (and/or a local proliferation of precursors), but also of the efflux of such cells. We investigated the recruitment of NOD monocytes, MΦ and DC into sites of acute inflammation in response to single chemoattractants, whereas the chronic autoimmune inflammations in the NOD mouse are the result of a range of inflammatory factors.

In conclusion, this is the first report that shows that NOD mice have an impaired ability to recruit infiltrating leukocytes into sites of artificially induced inflammation (peritoneal cavity and air pouch). To explain this phenomenon we favor the view that not only an increased IL-10/IL-1β ratio, but also a deficient migratory capacity of the monocytes of the NOD mouse plays a role. This defect is probably present in cells of the myeloid lineage, since granulocyte recruitment in NOD mice was also strongly reduced. Whether the impaired migration described here also applies to cells accumulating in the other autoimmune inflammations in the NOD mice is the focus of our current investigations.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Animals

Female NOD/LTj mice were bred in our facilities under specified pathogen-free conditions. Female C57BL/6 and BALB/c mice were obtained from Harlan (Horst, The Netherlands). All mice were fed standard pellets and received water ad libitum. The cumulative incidence of diabetes at 30 weeks of age in our NOD mouse colony was 90% in females and 60% in males. All animal procedures were carried out with the approval of the Erasmus University Animal Welfare Committee.

Peritonitis

Peritonitis was induced by intraperitoneal injection of sterile TG (3% w/v in 0.5 ml of sterile saline; Sigma, Steinheim, Germany). The mice were killed, at various time points, by carbon dioxide exposure, and peritoneal cavities were washed with 5 ml PBS containing 0.02% EDTA (Sigma). Cells were subsequently centrifuged (5 min/500×g), resuspended in 1 ml RPMI supplemented with 10% FCS (Biowhittaker, Verviers, Belgium) and kept on ice for further analysis.

Air pouch model

The air pouch model was applied as described elsewhere [17]. Briefly, pre-diabetic (5–7 weeks old) NOD and control C57BL/6 and BALB/c mice were subcutaneously injected on the back with sterile air (day 0: 5 ml; day 3: 3 ml). On day 6, 0.5 μg C5a (Sigma), CCL3 or CCL2 (Peprotech, London, UK) dissolved in 1 ml carboxymethylcellulose (Sigma; 0.5% in PBS, containing 10 ng/ml LPS) was injected into the air pouch. After 24 h, the animals were killed and the pouches were washed with 1 ml PBS. Volume and cell number of the lavage fluid was recorded and 100-μl aliquots were used for flow cytometry. The remaining fluid was centrifuged and the supernatant stored at –80°C for cytokine measurement.

To study the effect of IL-10, the air pouch was injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 μg/ml; R&D systems).

Flow cytometry

Flow cytometry was performed using a FACSCalibur apparatus (Becton Dickinson, Amsterdam, The Netherlands). The antibodies used were: biotinylated Ly-6C (ER-MP20; produced in our own laboratory), Ly-6G-PE, CD11b-PerCP-Cy5.5, CD11c-FITC, biotinylated CD11c (BD Pharmingen, Alphen aan den Rijn, The Netherlands) and F4/80-FITC (Caltag, San Francisco, CA). Streptavidin-APC (BD Pharmingen) was used as conjugate. For intracellular detection of IL-10, a biotinylated mAb against IL-10 (JES-2A5.1) and the isotype control GL113 were used (both obtained from BD Pharmingen).

In vitro migration assay

The in vitro migration towards CCL2 (Peprotech) of inflammatory peritoneal MΦ (TG-elicited, after 4 days) was evaluated using a Boyden chemotaxis chamber (Neuroprobe, Gaithersburg, MD) and polycarbonate membranes (5-μm pore size; Whatman, Clifton, NJ) as previously described. MΦ (3×106/ml) migration was determined after 4 h and expressed as a migration index (CCL2-migrated cells divided by the medium-migrated cells). Each experiment was performed in triplicate and cells were counted in five high-power fields (×1,000).

ELISA

The levels of IL-1β, IL-6 (R&D Systems, Minneapolis MN), IL-10, IL-12 and TNF-α (BioSource, Camarillo CA) were measured in air pouch exudates using ELISA, according to the manufacturers protocol.

Statistics

Data were analysed using Student's t-test or Mann-Whitney. When more than two groups were compared, Kruskall-Wallis test was performed, followed by Mann-Whitney test to determine the significance of the differences between the individual groups. All data were analyzed for two-tailed significance. A p value below 0.05 was considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This study was supported by research grants from the European Union QLRT-1999–00276 (MONODIAB) and the Dutch Diabetes Research Foundation (96.606).

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 5

    WILEY-VCH

  • 6

    WILEY-VCH

  • 7

    WILEY-VCH

  • 8

    WILEY-VCH

  • 9

    WILEY-VCH

  • 1
    Jansen, A., Homo-Delarche, F., Hooijkaas, H., Leenen, P. J., Dardenne, M. and Drexhage, H. A., Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and beta- cell destruction in NOD mice. Diabetes 1994. 43: 667675.
  • 2
    Rosmalen, J. G., Homo-Delarche, F., Durant, S., Kap, M., Leenen, P. J. and Drexhage, H. A., Islet abnormalities associated with an early influx of dendritic cells and macrophages in NOD and NODscid mice. Lab. Invest. 2000. 80: 769777.
  • 3
    Gagnerault, M. C., Luan, J. J., Lotton, C. and Lepault, F., Pancreatic lymph nodes are required for priming of beta cell reactive T cells in NOD mice. J. Exp. Med. 2002. 196: 369377.
  • 4
    Springer, T. A., Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994. 76: 301314.
  • 5
    Zlotnik, A. and Yoshie, O., Chemokines: a new classification system and their role in immunity. Immunity 2000. 12: 121127.
  • 6
    Gerszten, R. E., Garcia-Zepeda, E. A., Lim, Y. C., Yoshida, M., Ding, H. A., Gimbrone, M. A., Jr., Luster, A. D., Luscinskas, F. W. and Rosenzweig, A., MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature 1999. 398: 718723.
  • 7
    Hurst, S. M., Wilkinson, T. S., McLoughlin, R. M., Jones, S., Horiuchi, S., Yamamoto, N., Rose-John, S., Fuller, G. M., Topley, N. and Jones, S. A., IL-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation. Immunity 2001. 14: 705714.
  • 8
    Dustin, M. L., Rothlein, R., Bhan, A. K., Dinarello, C. A. and Springer, T. A., Induction by IL 1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J. Immunol. 1986. 137: 245254.
  • 9
    Mantovani, A. and Dejana, E., Cytokines as communication signals between leukocytes and endothelial cells. Immunol. Today 1989. 10: 370375.
  • 10
    Frosch, M., Strey, A., Vogl, T., Wulffraat, N. M., Kuis, W., Sunderkotter, C., Harms, E., Sorg, C. and Roth, J., Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis. Arthritis Rheum. 2000. 43: 628637.
  • 11
    Macatonia, S. E., Hosken, N. A., Litton, M., Vieira, P., Hsieh, C. S., Culpepper, J. A., Wysocka, M., Trinchieri, G., Murphy, K. M. and O'Garra, A., Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J. Immunol. 1995. 154: 50715079.
  • 12
    Randolph, G. J., Beaulieu, S., Lebecque, S., Steinman, R. M. and Muller, W. A., Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 1998. 282: 480483.
  • 13
    Fiorentino, D. F., Zlotnik, A., Mosmann, T. R., Howard, M. and O'Garra, A., IL-10 inhibits cytokine production by activated macrophages. J. Immunol. 1991. 147: 38153822.
  • 14
    Shrikant, P., Lee, S. J., Kalvakolanu, I., Ransohoff, R. M. and Benveniste, E. N., Stimulus-specific inhibition of intracellular adhesion molecule-1 gene expression by TGF-beta. J. Immunol. 1996. 157: 892900.
  • 15
    Melnicoff, M. J., Horan, P. K. and Morahan, P. S., Kinetics of changes in peritoneal cell populations following acute inflammation. Cell. Immunol. 1989. 118: 178191.
  • 16
    Edwards, J. C., Sedgwick, A. D. and Willoughby, D. A., The formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system. J. Pathol. 1981. 134: 147156.
  • 17
    Romano, M., Sironi, M., Toniatti, C., Polentarutti, N., Fruscella, P., Ghezzi, P., Faggioni, R., Luini, W., van Hinsbergh, V., Sozzani, S., Bussolino, F., Poli, V., Ciliberto, G. and Mantovani, A., Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity 1997. 6: 315325.
  • 18
    Sunderkotter, C., Nikolic, T., Dillon, M. J., van Rooijen, N., Stehling, M., Drevets, D. A. and Leenen, P. J. M., Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J. Immunol. 2004. 172: 44104417.
  • 19
    Geissmann, F., Jung, S. and Littman, D. R., Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 2003. 19: 7182.
  • 20
    Demangel, C., Bertolino, P. and Britton, W. J., Autocrine IL-10 impairs dendritic cell (DC)-derived immune responses to mycobacterial infection by suppressing DC trafficking to draining lymph nodes and local IL-12 production. Eur. J. Immunol. 2002. 32: 9941002.
  • 21
    Fine, J. S., Rojas-Triana, A., Jackson, J. V., Engstrom, L. W., Deno, G. S., Lundell, D. J. and Bober, L. A., Impairment of leukocyte trafficking in a murine pleuritis model by IL-4 and IL-10. Inflammation 2003. 27: 161174.
  • 22
    Lynch, D. M. and Kay, P. H., Studies on the polymorphism of the fifth component of complement in laboratory mice. Exp. Clin. Immunogenet. 1995. 12: 253260.
  • 23
    Leon, B., Martinez del Hoyo, G., Parrillas, V., Vargas, H. H., Sanchez-Mateos, P., Longo, N., Lopez-Bravo, M. and Ardavin, C., Dendritic cell differentiation potential of mouse monocytes: monocytes represent immediate precursors of CD8 and CD8+ splenic dendritic cells. Blood 2004. 103: 26682676.
  • 24
    Strid, J., Lopes, L., Marcinkiewicz, J., Petrovska, L., Nowak, B., Chain, B. M. and Lund, T., A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse. Clin. Exp. Immunol. 2001. 123: 375381.
  • 25
    Serreze, D. V., Gaskins, H. R. and Leiter, E. H., Defects in the differentiation and function of antigen presenting cells in NOD/Lt mice. J. Immunol. 1993. 150: 25342543.
  • 26
    Morel, P. A., Vasquez, A. C. and Feili-Hariri, M., Immunobiology of DC in NOD mice. J. Leukoc. Biol. 1999. 66: 276280.
  • 27
    Faveeuw, C., Gagnerault, M. C. and Lepault, F., Expression of homing and adhesion molecules in infiltrated islets of Langerhans and salivary glands of nonobese diabetic mice. J. Immunol. 1994. 152: 59695978.
  • 28
    Bonita, R. E., Rose, N. R., Rasooly, L., Caturegli, P. and Burek, C. L., Adhesion molecules as susceptibility factors in spontaneous autoimmune thyroiditis in the NOD-H2 h4 mouse. Exp. Mol. Pathol. 2002. 73: 155163.
  • 29
    Josefsen, K., Nielsen, H., Lorentzen, S., Damsbo, P. and Buschard, K., Circulating monocytes are activated in newly diagnosed type 1 diabetes mellitus patients. Clin. Exp. Immunol. 1994. 98: 489493.
  • 30
    Hill, H. R., Augustine, N. H., Rallison, M. L. and Santos, J. I., Defective monocyte chemotactic responses in diabetes mellitus. J. Clin. Immunol. 1983. 3: 7077.
  • 31
    Canning, M. O., Grotenhuis, K., De Haan-Meulman, M., De Wit, H. J., Berghout, A. and Drexhage, H. A., An abnormal adherence of monocytes to fibronectin in thyroid autoimmunity has consequences for cell polarization and the development of veiled cells. Clin. Exp. Immunol. 2001. 125: 1018.
  • 32
    Kuijpens, J. L., De Hann-Meulman, M., Vader, H. L., Pop, V. J., Wiersinga, W. M. and Drexhage, H. A., Cell-mediated immunity and postpartum thyroid dysfunction: a possibility for the prediction of disease? J. Clin. Endocrinol. Metab. 1998. 83: 19591966.
  • 33
    Bouma, G., Lam-Tse, W. K., Wierenga-Wolf, A. F., Drexhage, H. A. and Versnel, M. A., Increased serum levels of MRP-8/14 in type 1 diabetes induce an increased expression of CD11b and an enhanced adhesion of circulating monocytes to fibronectin. Diabetes 2004. 53: 19791986.
  • 34
    Naito, M., Hasegawa, G. and Takahashi, K., Development, differentiation, and maturation of Kupffer cells. Microsc. Res. Tech. 1997. 39: 350364.
  • 35
    Wu, L., Li, C. L. and Shortman, K., Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. J. Exp. Med. 1996. 184: 903911.
  • 36
    Melnicoff, M. J., Horan, P. K., Breslin, E. W. and Morahan, P. S., Maintenance of peritoneal macrophages in the steady state. J. Leukoc. Biol. 1988. 44: 367375.