Peyer's patch organogenesis—cytokines rule, OK?

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  1. GRAHAM MAYRHOFER
  1. Acquaintance Professor, Department of Microbiology and Immunology, Academy of Adelaide and Arthritis Research Laboratory, Hanson Centre for Cancer Research, Institute of Medical and Veterinarian Science, Adelaide, Southward Australia

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Information technology seems that wherever ane turns in mammalian biological science, from formulation through normal development to death, cytokines of immunological interest accept become inserted every bit important regulators of tissue activeness. Or have they? The truth is that at various stages the evolving immune organisation has borrowed widely to turn existing regulatory and effector molecules to new advantages.

The tumour necrosis factor (TNF) and TNF receptor (TNFR) families

The TNF and TNFR families of molecules1-iii consist of the ligands TNF, lymphotoxins alpha and beta (LT-α, LT-β), nerve growth factor (NGF), Fas ligand (FasL), CD27 ligand, CD30 ligand, CD40 ligand, OX40 (CD134) ligand, and 4–1BB ligand and their receptors (which, in almost cases, are ligand specific). Of the ligands, TNF, LT-α and NGF are secreted products, which in each ease acquaintance to course biologically agile homotrimers (LT-α3 in the instance of LT-α). Both TNF and LT-α3 are approximately every bit favoured ligands for 2 receptors, TNFR I (55 kD) and TNFR II (75 kD). LT-β is a membrane protein that is not secreted and has non been observed as a homotrimer. Expression of LT-β on the cell surface seems to require co-expression of LT-α and their association in the heterotrimers LT-α2β1 (minority) or LT-α1βtwo (majority). LT-α2β1 may signal via either of the TNF receptors only LT-αoneβ2 signals via a specific receptor (LTβ-R; likewise known as TNFR related protein, TNFRrp).

The TNF ligand family are, in general, inducers of either prison cell proliferation or cell expiry and the receptors for some are expressed by a wide range of tissues. It would not, therefore, take been surprising if loss of function in ligand/receptor pairs in this family by natural mutations or through gene targeting had caused significant defects in general embryonic morphogenesis. In fact, this has not been the case, fifty-fifty when a double knockout of the genes encoding TNF-α and LT-α has prevented all signalling past these cytokines or past LT-β (table ane). However, loss of office in genes encoding the ligand/receptor pairs FasL/Fas; TNF-α, LT-α/TNFR I and LT-α,β/LTβ-R is accompanied by morphological abnormalities that are limited to the lymphoid tissues. In the cases of TNF-α and LT, information technology is uncertain whether the morphological changes are primarily defects in morphogenesis or whether they are secondary to functional deficiencies in the behaviour of various differentiated cells of the lymphoid system (as seems to be the case in natural mutations of either Fas or FasL).

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Table 1

 Predicted activities of TNF/LT ligands in factor knockout mice

Organogenesis of Peyer's patches in mice lacking TNF-α or the TNFR I

A recent publication by Pasparakis and colleagues examines the gross anatomical, histological and cellular abnormalities that follow targeted disruption of the genes encoding TNF-α and 1 of its receptors, TNFR I (p55TNF-R). From the foregoing discussion (table 1) information technology can be seen that TNF-α−/− and TNFR I−/− mice need not exhibit equivalent phenotype because the TNFR I also serves LT-α. Both homozygous knockout mutants (on mixed 129Sv X C57BL/6 background) had normal thymic morphology and function, grossly normal lymph nodes (LN) merely had Peyer's patches (PP) that were reduced both in size and number. Peyer's patches were flat, they appear from the illustrations to lack subdivision into distinct follicles, while baloney of normal morphology (lack of clear follicle associated epithelium and organised patch associated villi) was more than axiomatic in the TNFR I−/− mice. Nevertheless, the PP contained both B and T cells and in that location was some segregation of T cells into small-scale presumptive interfollicular areas. Dendritic cells were present within these concentrations of T cells and besides within the rudimentary dome areas, an interesting observation in low-cal of the importance of TNF-α in the maturation of dendritic cells and their migration to lymph nodes.4 On the other hand, in both PP and LN, too equally in spleen,5 at that place was a failure of B lymphocytes to organise into primary follicles, a failure of germinal heart development and an absenteeism of follicular dendritic cell (FDC) networks within areas of B cells.

Pasparakis et al believe that the main defect in all of the secondary lymphoid tissues in their mice lies downstream of primary morphogenesis and is related more to a role of TNFα/TNFR I in the migration/organisation of B cells to grade follicular structures. In particular, they favour a key role for this ligand/receptor pair in the differentiation or arrangement, or both, of FDC into a network effectually which the follicles can develop. If right, information technology is a fine point equally to whether such a defect is morphogenetic (ie, failure of mesenchyme to commit to the FDC lineage during embryogenesis) or functional (failure of FDC to respond to signalling through TNFR I) and it is relevant that FDC express TNFR I and too make TNF-α.6 It is as well legitimate to question whether a reduction in PP number does not, at face up value, indicate at least a partial failure of morphogenesis. It is noteworthy that initial reports on PP phenotypes in TNF−/− mice5 and TNFR I−/−mice have been revised.seven ,viii Later on initial reports in which it was stated that PP were absent-minded in both gene targeted mutants, it seems now that in each case, PP were in fact present although small and difficult to place. This conveys a articulate bulletin that in whatever mutation that affects the evolution of PP, anything less than complete series sections of the small intestine would neglect to provide convincing testify that small anlagen accept not been missed and that PP morphogenetic foci are not present in normal numbers. It is not clear from the studies on TNF-α−/− or TNFR I−/− mice whether failure to signal via the TNFR I (past TNF and/or LT-α) reduces the number of anlagen (ie, is important for the initiation of the correct number of anlagen) or whether its role lies downstream and affects the subsequent development of those anlagen. Furthermore, the analysis to engagement is too crude to permit a conclusion that the defects in the TNF-α−/− and TNFR I−/− mice are equivalent and therefore, that LT-α3 and LT-αiβii produce no PP phenotype.

A contempo study on an independent TNF-α−/− knockout, this time performed in C57BL/6 mice, is informative on three counts.9 Firstly, at least in spleen, areas of B cells do contain scattered cells stained by monoclonal antibody FDC-M1, indicating that FDC indeed differentiate in TNF-α−/−mice and that their absence is not the primary defect in PP morphogenesis in these animals (their functional country is another matter). Secondly, in this mouse strain there was only a small divergence in the numbers of PP betwixt TNF-α−/− and wild type mice, although morphologically they resembled the structures described by Pasparakis et al. This observation suggests that TNF-α is non a primary morphogenetic stimulus for the evolution of PP. It raises a further question: do morphogenetic events lay down a template for coordinated epithelial differentiation (follicle associated and patch villus associated) and is this at least partially nether the command of TNF-α, or does subsequent lymphoid development and function shape the epithelial architecture and is this the mode that TNF-α exerts its result on PP morphology? My ain response to this question is to enquire which is the most unique feature of a PP. To me, it is the follicle associated epithelium and past this reasoning, my speculation is that principal organisers in PP organogenesis volition create focal fields of epithelial differentiation, which volition in turn induce differentiation of underlying mesenchyme. Finally, Körner et al 9 observed that the PP "homing receptor" MadCAM was expressed by vessels in PP. This supports the contention of Pasparakis et al that failure of lymphocyte entry into PP is not responsible for the PP abnormalities in TNF-α−/− mice. Körner et al 9 conclude that early morphogenesis of PP is controlled mainly by LT (meet later on), whereas the function of TNF-α is related more than to the organization of lymphocytes within the edifice then created. Information technology would be of interest to know whether PP morphology in TNF-α−/− mice could be restored to normal by adoptive transfer of recirculating mature lymphocytes (ie, whether the defect lies in the response of stromal components of the PP or of lymphocytes to signalling via TNF-α.)

The importance of lymphotoxins in organogenesis of Peyer's patches

A more than severe phenotype would be expected by disruption of genes which impinge more than globally on the functions of the TNF/LT family of ligands (table 1). Targeting the TNFR I gene (TNFR Ii does not seem to exist relevant in the context of this discussion) can block the deportment of TNF-α, LT-αthree and LT-αoneβ2but signalling can still occur via membrane bound LT-α1β2. Peyers patches in TNFR I−/− mice seem to be reduced in number8and they have more severe disturbance of compages than those in TNF-α−/− mice (Pasparakis et al).8 LT-α3 and/or LT-α2β1 may, therefore, have a function in the germination of normal PP, but they practice not seem to be the primary organisers of PP anlagen. If all membrane spring LT activity is abolished (LT-α−/− mice), there is a striking phenotype, with agenesis of PP and all LN, disrupted splenic white pulp architecture but normal thymic development. There seems no question simply that membrane leap LT is vital in the organogenesis of secondary lymphoid organs, including PP. This conclusion is borne out (almost completely) past studies in LT-β−/− mice, where at that place is selective loss of membrane associated LT. These mice lack PP and virtually LN but they do have mesenteric LN and variable numbers of cervical LN (albeit with abnormal structure). As might be expected, the coup-de-grâce (elimination of both TNF-α and LT-α) also leads to agenesis of PP and all LN.ix ,10

It is interesting that lymph nodes vary in their requirements for LT-αiβ2 to induce organogenesis. A fascinating addition to this story is the utilise of maternally administered LTβ-R-Ig fusion protein to block membrane bound LT at various stages of gestation within the fetus.11Administration of fusion protein every bit early on equally 9 days' gestation did not inhibit morphogenesis of mesenteric LN, whereas development of other LN was blocked even if fusion protein treatment was delayed until day 12 (brachial LN) through to solar day 16 (popliteal LN). In contrast, morphogenesis of PP was prevented even when handling was given on mean solar day xviii of gestation. These results correlate with the late maturation of the gut in rodents and the appearance of PP anlagen in the late prenatal menses.12 More needs to be washed to examine the precise time at which genes that encode TNF-α, LT-α, LT-β, and their receptors are transcribed in the local mesenchyme that gives ascent to LN and PP.

IgA production in gene targeted mice

Mice with selectively targeted genes encoding LT-α13 ,14 or LT-β14 or with combined knockouts of the genes encoding TNF-α and LT-α9 ,10provide a unique opportunity to assess the importance of PP for IgA production (although other sites, such as bronchus associated lymphoid tissue, could also be affected). LT-α−/− and LT-β−/− mice take very depression concentrations of IgA in serum and in faeces, although serum concentrations of IgM and IgG are relatively normal. Mesenteric LN, present in LT-β−/−mice only not in LT-α−/− mice, do non seem to confer a greater capacity to produce IgA. The presence of normal concentrations of serum IgG in these mice suggests that the defect in IgA product results from agenesis of PP rather than to a more general failure to support isotype switching. In TNF-α/LT-α−/− double knockout mice, serum IgA and mucosal IgA plasma cells are even more profoundly depressed and in that location is also reduction in serum IgG concentrations.9 ,x These findings may indicate that some switching to the IgA isotype can occur at other sites (eg, the spleen) in LT-α−/− and LT-β−/− mice.

In contrast, the structural disorganisation in PP of TNF-α−/− mice does not pb to reduction in serum IgA concentrations.9 The same seems to exist true for TNFR I−/− mice, which take been reported to lack PP8 but seem now to have pocket-size and disorganised PP (Pasparakis et al).

Implications for mucosal immunologists and gastroenterologists

What lessons tin can be learned from factor targeted mice with abnormal or absent-minded PP? Firstly, the mutants in which PP are affected (relatively) selectively offer opportunities for exploring the roles of these organs in responses to immunising and tolerising antigens delivered orally. Secondly, the mutants with accented agenesis of PP offer an interesting model in which to explore the permeability of the villous mucosa to antigens and macromolecules, independent of PP (and, presumably, isolated lymphoid follicles). Thirdly, how do the results in rodents relate to humans, in whom information technology is reported that numbers of PP increase until puberty?15 Does this represent delayed morphogenesis de novo, or does it represent the activation of latent anlagen (perhaps by the activeness of cytokines such as TNF-α)? Do PP that involute in later life disappear, or can they be reactivated? Our ain recent studies suggest that a range of isolated lymphoid aggregations that are present in normal man small intestine can exist activated by microbial colonisation. Finally, how practise these findings on early morphogenesis relate to the "lymphoid neogenesis" observed in transgenic mice when LT-α is expressed ectopically under a tissue specific promoter?16 Is follicular hyperplasia in the pocket-size intestine, follicle germination in the breadbasket in atrophic gastritis or the mononuclear infiltrate in Crohn's disease a recapitulation of morphogenetic events that occur unremarkably under control of cytokines during early on development? Love them or hate them, cytokines rule.

Acknowledgments

The author cheers Dr Jonathon Sedgwick, Centenary Found for Cancer Medicine and Jail cell Biology (Sydney) for access to Körneret al's (1997) manuscript.

References

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Pasparakis M , Alexopoulou 50 , Grell M , Pfizenmaier G , Bluethmann H , Kollias Yard (1997) Peyer'due south patch organogenesis is intact nevertheless germination of B lymphocyte follicles is lacking in peripheral lymphoid organs of mice deficient for tumor necrosis factor and 55-kDa receptor. Proc Natl Acad Sci USA.

Targeted inactivation of genes in the tumor necrosis factor (TNF)/lymphotoxin (LT) ligand and receptor system has recently revealed essential roles forthese molecules in lymphoid tissue development and organisation. Lymphotoxin-αβ (LTαβ)/lymphotoxin-β receptor (LTβ-R) signaling is critical for the organogenesis of lymph nodes and Peyer's patches and for the structural compartmentalization of the splenic white pulp into singled-out B and T jail cell areas and marginal zones. Moreover, an essential function has been demonstrated for TNF/p55 tumor necrosis factor receptor (p55TNF-R) signaling in the formation of splenic B lymphocyte follicles, follicular dendritic jail cell networks, and germinal centers. In contrast to a previously described essential office for the p55TNF-R in Peyer's patch organogenesis, we show in this report that Peyer's patches are nowadays in both TNF and p55TNF-R knockout mice, demonstrating that these molecules are not essential for the organogenesis of this lymphoid organ. Furthermore, we show that in the absence of TNF/p55TNF-R signaling, lymphocytes segregate ordinarily into T and B cell areas and a normal content and localization of dendritic cells is observed in both lymph nodes and Peyer's patches. However, although B cells are found to abode normally within Peyer'southward patches and in the outer cortex area of lymph nodes, organized follicular structures and follicular dendritic prison cell networks neglect to form. These results show that in contrast to LTαβ signaling, TNF signaling through the p55TNF-R is non essential for lymphoid organogenesis but rather for interactions that determine the cellular and structural organization of B cell follicles in all secondary lymphoid tissues.

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