The abluminal crawling of neutrophils

During the inflammatory reaction to an invading pathogen neutrophils arrive at the injury site and leave blood vessels to accumulate around foreign particles. Leukocytes exiting the bloodstream have to breach first through endothelial cells before they enter the interstitial space. The publication I am discussing today adds yet another step to this route as it analyzes how neutrophils migrate through the layer of pericytes that coats endothelial cells. Pericytes are cells that form the important structural part of certain types of blood vessels (capillaries, postcapillary venules, and collecting venules); however, their participation in leukocyte passage was previously unstudied. This paper extends the knowledge of neutrophil migration to the inflamed tissue by describing the novel type of movement coined abluminal crawling and exploring many intricacies of neutrophils/pericytes interactions. My experience in the main technique applied by authors – the real-time in vivo imaging – is next to nothing, but I can still admire how they put to use their skills.

The link: http://jem.rupress.org/content/209/6/1219.abstract

Investigators visualize pericytes and neutrophils by generating the mouse strain that has fluorescent proteins expressed in the above cell types. Additionally, endothelial cells are made visible with the in vivo application of specific immuno-staining.  The migration of neutrophils to peripheral tissue is provoked by the injection of potent inflammatory agent (TNF) into the exposed body part of such mice (cremasteric muscle located in the scrotum). Authors observe that after the rapid breach through endothelial cells neutrophils display prolonged association with the pericyte layer combined with the movement along pericyte processes – the already mentioned abluminal crawling. The abluminal crawling ends when a neutrophil leaves into the interstitial space through a gap between pericytes.

How the abluminal crawling is regulated? The efficient neutrophil movement along pericytes depends on the expression of ICAM-1 (on pericytes) as well as Mac-1 and LFA-1 (on neutrophils). The average gap size between pericytes (which is proposed by investigators to be one of decisive factors underlying successful migration) is enlarged by the injection of proinflammatory cytokines TNF and IL-1β and pericytes express receptors recognizing these mediators. Authors also show the intriguing data suggesting that the gap choice by neutrophils is apparently not random as very often a single gap is used by multiple neutrophils. To sum up, this report implicates that pericytes may participate in the immune response by influencing the neutrophil migration into periphery.

Proebstl D, Voisin MB, Woodfin A, Whiteford J, D’Acquisto F, Jones GE, Rowe D, & Nourshargh S (2012). Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo. The Journal of experimental medicine, 209 (6), 1219-34 PMID: 22615129

Neutrophils, IL-17 and oral microbiota in periodontitis

Th17 response has the ability to induce migration of neutrophils from the bloodstream into inflamed tissues. Neutrophils form the important part of immune defense and as such are equipped with a number of anti-microbial and pro-inflammatory measures. However, as in the case of other immune effectors their action may also have the darker side – collateral injuries to the surrounding environment. Such immune-mediated bystander damages underlie the pathology of periodontitis which is the inflammatory disease afflicting gingival tissue. I have read very interesting publication that describes how the intricate interplay between neutrophils and IL-17 cytokine may regulate periodontitis development.

The link: http://www.nature.com/ni/journal/v13/n5/full/ni.2260.html

Periodontitis is a disease that inflicts neutrophil-mediated inflammatory lesions to the tooth-supportive tissue which may result in loosening and loss of teeth. Old mice just like old humans are prone to develop periodontitis. Del -1 is a negative regulator of neutrophil extravasation which is known to be expressed by endothelial cells (cells that line blood vessels). Authors initially explored Del-1 expression pattern in various age groups as the association between the age and the disposition to excessive inflammation is well known. They concluded that Del-1 level in the gingival tissue of older mice represented only a small portion (~25%) of Del-1 amount found in younger mice. They also found that the decrease in Del-1 expression correlated with more pronounced tooth bone loss and the enhanced neutrophil influx to gingiva. The Del-1/neutrophils/bone loss relationship was confirmed by using Del-1 deficient mice. Apart from that it was shown that the increase in gingival neutrophil infiltration mediated by the deletion of Del-1 could be counteracted by knocking out LFA-1 which is a positive regulator involved in neutrophil tissue migration.

Del-1 deletion seemed to augment the local Th17 response as Del-1 deficient mice expressed more IL-17A (IL-17C and IL-17F were also increased) as well as p40 and p19 (subunits of IL-23 which is strong Th17 response inducer). Remarkably, the analysis of IL-17RA-deficient mice has shown that inflammatory bone loss characteristic for periodontal injuries was completely abolished when there was no IL-17 signaling (interestingly, there was no difference between IL17RA-deficient strain and combined IL-17RA/Del-1 mutant). IL-17RA deletion also enhanced Del-1 expression in gingival tissues. Investigators confirmed the link between lack of IL-17 signaling and increase in Del-1 amount by neutralization method (injection of monoclonal anti-IL17 antibody to gingiva) and bone marrow chimeras experiments. The last approach revealed that the lack of IL-17RA on non-hematopoietic cells was important in regulating Del-1 expression. Finally, authors explored whether administration of Del-1 into inflamed gingival tissues could have the therapeutic potential

Beyond results discussed above this publication contains data on how genetic background that underlies quantitative aspects of neutrophil migration into gingiva may influence the oral microbiota. These data are somewhat counter-intuitive (as it is sometimes the case for things happening at mucosal surfaces). The leading theme of this paper is that Del-1 down-regulation or absence promotes the enhanced neutrophil infiltration and augments inflammatory-mediated tooth bone loss. However, Del-1 deletion (and hence more gingival neutrophils, which after all are thought to be cells with anti-microbial properties) actually stimulates more bacterial growth. This not just one odd result, because when Del-1 deletion is combined with LFA-1 knockout (a positive regulator of neutrophil extravasation) or IL-17RA knockout (removing thus the important part of signaling involved in the neutrophil influx) the number of oral anaerobic bacteria goes back to normal values. Thus to sum up, the excessive inflammation seems to boost but not to restrict the growth of oral microbiota. Authors do not follow this observation but I think it is very interesting phenomenon.

Eskan, M., Jotwani, R., Abe, T., Chmelar, J., Lim, J., Liang, S., Ciero, P., Krauss, J., Li, F., Rauner, M., Hofbauer, L., Choi, E., Chung, K., Hashim, A., Curtis, M., Chavakis, T., & Hajishengallis, G. (2012). The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss Nature Immunology, 13 (5), 465-473 DOI: 10.1038/ni.2260

Neutrophils and marginal zone B cells – a new partnership

Being interconnected – that’s one of cardinal features pertinent to the mammalian immune system. Cellular subsets involved in an immune response seldom act alone beyond the initial signal recognition stage. Instead, the converging and augmentation of warning signs, the induction, adjustments or limitations of effector branches and finally the maintenance of immunological memory – these steps are usually carried out by two or more interacting populations. It is always so interesting to learn about new connections between cells of immune system, therefore recent report describing the supportive role of neutrophils in the antibody secretion process has immediately caught my attention.

The link to the publication:

http://www.nature.com/ni/journal/v13/n2/abs/ni.2194.html

This article reminds me the other, now pretty notorious story about the partnership of germinal center B cells and follicular helper CD4 T cells – an interaction that’s been proven to be so important during the antibody maturation process. But we have quite other players this time around.  Marginal zone B cells form the population posed at the anatomical intersection between circulation and lymphoid areas in the spleen. They are considered to possess blood filtering functions and are critically important in sepsis. There also were reports describing their pre-activated phenotype, which means that they can be easily stimulated to make antibodies. Neutrophils are most numerous granulocytes; they are cells extremely proficient in phagocytosis – kind of the always-ready emergency crew generally thought to be the first line of defense.

The research is performed on human cells combining imaging and functional data. The paper starts with series of pictures showing that neutrophils can colonize the splenic marginal zone under homeostatic but not inflammatory conditions. As a control authors perform the identical staining procedure with secondary lymphoid organs that have no marginal zone like tonsils, Peyer’s patches and peripheral lymph nodes. Then, compelling and diverse evidences are presented on how neutrophils enhance repertoire diversification and antibody secretion by marginal zone B cells. Apart from that, authors provide an in-depth description of the novel neutrophil “B helper” phenotype that is attained on contact with splenic epithelial cells.

However, it is the last and shortest paragraph of result section that really draws my attention. According to authors the increase in splenic colonization by neutrophils correlates with postnatal microbial colonization and fluorescence microscopy has shown LPS presence in adult but not fetal spleens. LPS was also shown to boost reprogramming of neutrophils to the “B helper” phenotype by splenic epithelial cells. Additionally, germ-free mice displayed lower levels of “B helper” neutrophils. Authors state – “Thus, we propose that TLR signals from mucosal commensals enhanced the splenic recruitment and reprogramming of N BH cells to enhance innate MZ B cell responses to highly conserved microbial TI antigens”.

Postnatal microbial colonization is the process of inhabiting intestines by commensal bacteria known as microbiota. It occurs soon after birth and has the instructive as well as regulatory effect on our immune system. Obviously the innate arm of immunity has the ability to detect microbial products and convey signals to start the inflammation. However, under normal circumstances the presence of microbiota is accommodated, although how exactly it happens is not entirely clear. Before I’ve read this paper I thought that so called microbial translocation or in other words the leakage of bacterial material from intestines into the bloodstream could happen exclusively as a pathological process.  Our body needs to be kept sterile; otherwise fatal disaster like sepsis may develop. So how to explain LPS presence in the spleen detected under homeostatic, non-inflammatory conditions? The most intuitive answer would be that the low-level trickling of unwanted bacterial stuff from the gut is taking place on the normal basis. Hence neutrophils may be assigned the duty of “garbage workers”, especially around big lymphoid organs like spleen.

I wish that somebody confirmed the neutrophils/marginal zone B cells partnership in murine models that may allow manipulation with relevant genetic backgrounds and cellular populations. The obvious problem to be solved is what will happen when there are no “B helper” neutrophils around. Will that impair the ability of marginal zone B cells to mount the effective protection against blood-borne antigens? However, it is not the only question I would ask. Authors present data showing that “B helper” neutrophils are able to contain CD4 T cells’ proliferation, although they never really explore this feature. May their function be more inhibitory than stimulatory? Somehow, that unexpected LPS presence in spleen keeps on intriguing me.  But now I have no idea how to interpret that.