A review of the functions and similarities of
Melanomacrophage centres and mammalian Germinal
centres and their use as a health indicator in fish
Killian Murray (s3601631)
Abstract
The health of fish in aquaculture farms is paramount for successful yields to be achieved
consistently with the need for a reliable indicator required to inform operators of any
potential harm occurring to the stock. A popular candidate for such a biomarker is
melanomacrophage centres (MMC), which are aggregates of pigmented cells involved in the
immune response of many vertebrates which are primarily found in the liver, spleen and
kidneys. Analysis indicates several similarities in structure and function between MMCs and
the mammalian germinal centre (GC). Being viewed as the evolutionary precursors to the
GC, there is considerable research to be conducted into how MMCs play into the adaptive
immune system with no outline as to how antigen affinity maturation occurs within the
hosts. The value of MMCs as a histological biomarker is high however it is necessary to
investigate further into these processes so that a greater understanding of how the MMC’s
role in the adaptive immune system was able to evolve into that of the GC’s role in
mammals. I suggest that a comparative gene expression study be conducted to determine
how MMC and GC function is similar and the exact method in which antibody affinity
maturation occurs within organisms without a GC. In the following review, a detailed
overview of the functions, both nonimmunological and immunological, are presented along
with the environmental factors that are involved in the optimum functioning of MMC.
Introduction
Aquaculture and the fishing industry as a whole contributed over $5 billion in 2017/18 to
the Australian economy (Fisheries Research and Development, 2019), and thus efficient
methods of determining fish health are required to ensure quality and protect quantity.
Infections, diet and other environmental conditions are known to cause changes in
melanomacrophage centres (MMCs) and thus extensive research has gone into their use as
a potential indicator of fish health.
MMCs are aggregates of pigmented cells that change with conditions that the fish is
subjected to in its environment. Pigmented with high concentrations of melanin, lipofuscin
and haemosiderin (Agius and Roberts, 2003), these cells are distinguishable by light
microscopy (Fig. 1) and are therefore, cost-effective and straightforward to survey. Whilst
locations within the organism are specific to species; they are most often found in
haematopoietic organs of fish including the spleen, liver and kidneys (Wolke, 1992). The
structure of MMCs in the liver and kidney are less defined than those in the spleen with the
latter being more organised and rigid (Falk, Press, Landsverk and Dannevig, 1995; Press,
Dannevig and Landsverk, 1994).
MMCs have also been found in species other than fish including amphibians and reptiles
however these have not received the same attention in literature (Steinel and Bolnick,
2017).
Figure 1: MMCs in spleen of stickleback (Gasterosteus aculeatus), (A) Unstained spleen slide at 50x magnification. (B) outlined in
box. (Scale bar = 250 μm). (B) Unstained spleen at 200x magnification. (C) Spleen stained with H&E at 200x magnification with
MMCs displayed with black arrows. (Scale bars for (B) & (C) =62.5 μm. (Steinel and Bolnick, 2017)
Nonimmunological functions of MMCs
The primary functions of MMCs are its involvement in both the innate and adaptive immune
system however they also have roles regarding the normal function of the body. First and
foremost, MMCs primarily conduct phagocytosis. Along with this, they have also been seen
as a storage site for unmetabolised waste products granting them the title of “metabolic
dump” (Agius, 1980).
Substances that are found within the aggregates can be categorised into two groups,
endogenous materials and exogenous materials (Agius, 1980). Endogenous materials
include melanin, lipofuscin and haemosiderin and the exogenous are further separated via
origin into experimental sources (carbon, polystyrene beads and antigens) and natural
sources (metals and biological agents) (Sayed and Younis, 2017). Of the endogenous
pigments found, only haemosiderin can be recycled while lipofuscin and melanin are stored,
with the latter potentially being used in the organism’s immune defence (Agius and Agbede
1984). Exogenous materials, particularly the experimental products, are stored in the MMC,
while natural sources are stored depending on the substance’s degradability.
Another largely documented function of MMCs is the recycling of iron and whilst there are
no direct experimental results, large amounts of circumstantial evidence indicate this to be
the case. Degenerating erythrocytes were observed in splenic MMCs as early as 1908
(Wolke, 1992). It is also known that aggregates of the spleen contain high quantities of
haemosiderin and when hyperhaemolytic crisis occurs, the pigment increased in
concentration and was found in MMCs of both liver and kidneys (Wolke, 1992). Finally, a
relationship between erythropoiesis and haemosiderin has been observed (Yu, Kiley, Sarot
and Perlmutter, 1971). Upon being injected with radio iron labelled erythrocytes from fish
into normal Trichogaster trichopterus, Yu et al. (1971) were able to measure and calculate
the proportion of iron that had settled in each organ. The spleen contained the highest
proportion of the metal featuring 55%. After stimulating erythropoiesis in 3 of 10 groups of
fish, no immediate changes were observed to the percentage of splenic MMC area occupied
by haemosiderin. Within 24 hours however it had dropped to 4.39 and 3.61 after 48 hours.
Neither liver nor kidneys featured any haemosiderin in the experimental animals with some
being present in the control animals.
Immunological functions of MMCs
Being formed by the aggregation of macrophages implies the role in phagocytosis. Whilst
erythrocytes are a known target of MMCs, they have been observed to phagocytose foreign
infectious materials (Brattgjerd and Evensen, 1996; Lamers. 1986). The aggregates of turtles
were observed as being highly aggressive and would seek out a variety of pathogens,
specifically bacteria, fungi and parasitic eggs (Johnson, Schwiesow, Ekwall and Christiansen,
1999). Being found in close proximity to the specialised capillaries called ellipsoids indicates
that MMCs may filter out pathogenic substances to then consume (Ferguson, 1976).
Studies conducted in vivo support this, finding that MMCs were able to quickly filter out
foreign substances injected into the host organism (Brattgjerd and Evensen, 1996).
The morphological similarities, along with being found in the same organs and their
association with infectious disease has led to the conclusion that MMCs are the
evolutionary precursor to germinal centres (GC) that are found in mammals (Fig. 2) (Agius
and Roberts, 2003). GCs are sites of antibody affinity maturation that are found in
secondary lymphoid organs (i.e. spleen and lymph nodes) (Allen, Okada and Cyster, 2007).
GCs allow antibodies to mutate to be more specific to the antigen being presented by the
Figure 2: Phylogeny showing vertebrates with and without MMC. This phylogeny is not historically to scale. (Steinel and Bolnick,
2017)
pathogen during the immune response (Lamers, 1986) and are where antigens are
processed for long term retention (Widdicombe et al., 2020). Unlike MMCs which are
aggregates of a singular type of cell, GCs are highly structured with multiple cell types,
featuring distinctive B cells, follicular dendritic cells (FDCs) and T follicular helper cell
aggregates (TFC). Following the presentation of antigens to the host, antigen specific B cells
accumulate and spread rapidly leading a temporary increase in GC size (Victora and
Nussenzweig, 2012). During the GC response (Fig. 3), B cells undergo clonal expansion and
differentiate into memory and plasma cells (Victora and Nussenzweig, 2012). FDCs are used
as antigen depots for future immunity by keeping them intact on their surface as immune
complexes (IC) storing them for recurrent infections. (Heesters, Myers and Carroll, 2014).
Antibody affinity maturation is a microevolutionary process in which antibodies undergo
somatic hypermutation (SHM), via the action of activation-induced cytidine deaminase (AID)
(Pavri and Nussenzweig, 2011). The GC B cells containing the now mutated antibodies are
then selected based on their affinity for the antigen (Berek, Berger and Apel, 1991).
Although teleosts do not have GCs, yet there are still able to generate an affinity matured
antibody response to antigen presentation (Solem and Stenvik, 2006). Studies across many
species of fish describe the numerous similarities between GCs and MMCs (Steinel and
Bolnik, 2017), raising the possibility that the aggregates are the site of adaptive immune
response in teleosts. Melanomacrophages (MM) stained positively for CNA-42 monoclonal
antibody (Vigliano, Bermúdez, Quiroga and Nieto, 2006) and express CSF1-R (Diaz-Satizabal
and Magor, 2015), both of which are found within in mammalian FDCs. When injected with
infectious and non-infectious materials, teleost MMCs were observed to be sites of antigen
retention (Lamers and De Haas, 1985; Herraez and Zapata, 1987). Antigens that are
retained are found near or within MMCs is extracellular or are trapped within ICs (Secombes
and Manning, 1980). Whilst this evidence linked MMs to FDCs found in the mammalian GC,
Figure 3: A diagram detailing the GC response. A specialised microenvironment which forms within B-cells after either
infection or immunisation. There are two sections, the dark zone (DZ) that contains a network of CXCL12-produing reticular
cells (CRCs) and is where GC B cell multiplication and SHM. Centroblasts then move into the light zone (LZ) as centrocytes
through the action of CXCR5. Centrocytes capture antigen from the FDC which they then present to the TFCs in order to be
selected. Centrocytes cycle back through the DZ for further rounds of proliferation and SHM (Stebegg et al., 2018)
other functions including phagocytosis and blood scavenging indicated a wider gamut of cell
types that the MMs act as in teleosts.
Infection and immunisation of teleosts have been shown to increase MMCs in size and/or
number in a similar manner to GCs (Agius, 1979; Herraez and Zapata, 1986; Widdicombe et
al., 2020). Lymphoid cells were shown to be in closely located to MMCs and upon
immunisation would increase (Herraez and Zapata 1986). No cell identity was discovered
due to a lack of species-specific reagents used. Recent studies were able to determine that
MMC adjacent immunoglobulin+ (Ig+) cells increase in size when experimentally infected
(Falk, Press, Landsverk and Dannevig, 1995; Press, Dannevig and Landsverk, 1994). While
assumed to be B cells, the Ig+ cells cannot be distinguished from cells expressing Ig and
those binding soluble Ig or IC to their surface (Falk, Press, Landsverk and Dannevig, 1995;
Press, Dannevig and Landsverk, 1994). Whilst some aggregation was observed amongst Ig+
cells, no highly compartmentalised structured was exhibited which are characteristic of
mammalian GC (Falk, Press, Landsverk and Dannevig, 1995; Press, Dannevig and Landsverk,
1994). As stated prior, GCs response leads to an increase in antibody production, something
not true in the MMC response (Herreaz and Zapato, 1986). AID expressing cells have been
more recently observed as being in or proximal to MMCs (Saunders et al., 2010). Due to the
requirement of AID expression for SHM to occur in GC, this would strongly support
hypothesis of a GC-like MMC. However, no SHM has been documented in MMC (Saunders
et al., 2010). These studies display a strong connection between GCs and MMCs with them
likely performing similar functions but do display functional differences as well.
Environmental factors and MMCs
Environmental factors can cause stress in organisms that leads to a susceptibility to
infection. Along with changes to the numbers of lymphocyte and granulocytes circulating,
the greatest changes are the abundance of macrophage cells, a reduction in the numbers of
platelets and lymphocytes and an increase in red blood cell degradation (Peters and
Schwarzen, 1985). When living in polluted environments, teleosts come to reflect the lesser
conditions through altered immune system activity (Kelly-Reay and Weeks-Perkins, 1994).
The size of MMCs found in the kidney and spleen of Southern Bluefin Tuna have been
observed to increase when the tuna is infected with Cardicola spp (Widdicombe et al.,
2020). Whilst MMCs have been known to change in size with age, their changes with
respect to their environment have led to the suggestion of them for use as biomarkers as a
measure of pollutants both biotic and abiotic.
Several studies have been conducted for histological indicators of environmental quality.
Pollutants, while potentially being fatal, sublethal effects are more common (Blazer, Wolke,
Brown and Powell 1987). Contaminated waters have been proven to increase the
prevalence of parasites and various other diseases in multiple marine fish populations
(Haaparanta, Valtonen, Hoffmann and Holmes 1996). A range of physiological and
pathological responses have been posited for use as markers including incidences of tumour
formation in epidermis and liver (Fabacher and Baumann 1985) and the presence of
xenobiotic-metabolising enzymes being found in particularly sensitive species (Stegeman
1978). The issue with these responses is that they are too specific to the substance that
triggers them and thus cannot be used as a generalised indicator.
MMC were identified by several authors as being apart of many disease processes and the
alterations that coincide with them including starvation (Agius and Roberts, 1980) or
chemical exposure (Meinelt, Kruger, Pietrock, Osten & Steinberg 1997) indicates that these
centres can be used as a sensitive indicator of stressful conditions in aquatic environments
(Agius and Roberts, 2003; Wolke, Murchelano, Dickstein and George, 1985). They are found
in close to all fish species, easily measured and able to be compared statistically.
Studies comparing MMC prevalence in different organs in fish extracted from water
polluted with toxic chemicals have seen contradicting results. Some authors observed an
increase in number and size (Wolke, Murchelano, Dickstein and George, 1985) while others
have recorded the opposite occurring (Kranz, 1989). The latter should be noted that at low
levels of pollution, an increase was observed and was attributed to the cellular defence
system to remove debris through an increase in phagocytic activity which was followed by
an increase in MMCs.
Further Research
In order to determine whether MMCs function in the same manner as GCs, further studies
are required. A possible method of this would be to determine whether the MMC is
capable of specific B cell clonal expansion and high-antibody affinity production, both of
which are the results of the GCs B cell proliferation, differentiation and SHM. While the
presence of AID-expressing cells in MMCs has led to the implication of that antibody affinity
maturation occurs (Saunders et al., 2010), more study into whether the action of antibody
SHM gene within and is dependent on the MMC is required. In the GC, the response is Tcell dependent as the development of B-cells requires the assistance of TFC. T-cell
dependent antigens are proteins that are required to induce the GC response whereas T-cell
independent antigens are polysaccharide based and therefore cannot be presented in TFC
or induce the GC response (Fagarasan, 2000). In order to determine the degree of similarity
between the MMC and GC response, a study into the effect of both T cell dependent and
independent antigens on the MMC response would be useful. Another avenue of research
could be a comparative analysis of MMCs and GCs to determine the similarities and
differences in gene expression. By observing these comparisons, one would be able to
provide more clarity as to whether the GC is evolutionary connected to the MMC.
Conclusions
The MMC shares several similarities with the mammalian GC including structural, cellular
and molecular, implying an evolutionary link between the two and the MMCs role in the
adaptive immune system. It is due to the parallels between the two centres that they have
been used as a favoured histological biomarker. I believe that while there is more research
to be conducted to determine the similarity between the action of MMC and GC, MMCs
have been proven as a reliable histological biomarker. It is important that research
continues, and should the findings validate MMCs as an immunological tool, studies
surrounding the health of aquaculture stock can potentially improve the quality of fish and
decrease the cost of stock maintenance. Clarity around the evolution of MMCs and GCs will
also improve, providing a greater understanding of the changes the immune system has
undergone.
Reference:
Agius, C., 1979. The role of melano-macrophage centres in iron storage in normal and
diseased fish. Journal of Fish Diseases, 2(4), pp.337-343.
Agius, C., 1980. Phylogenetic development of melano-macrophage centres in fish. Journal of
Zoology, 191(1), pp.11-31.
Agius, C. and Agbede, S.A. (1984). An electron micro- scopical study on the genesis of
lipofuscin, melanin and haemosiderin in the haemopoietic tissues of fish. Journal of Fish
Biology 24: 471-488.
Agius, C. and Roberts, R., 1981. Effects of starvation on the melano-macrophage centres of
fish. Journal of Fish Biology, 19(2), pp.161-169.
Agius, C. and Roberts, R., 2003. Melano-macrophage centres and their role in fish
pathology. Journal of Fish Diseases, 26(9), pp.499-509.
Allen, C., Okada, T. and Cyster, J., 2007. Germinal-Center Organization and Cellular
Dynamics. Immunity, 27(2), pp.190-202.
Berek, C., Berger, A. and Apel, M., 1991. Maturation of the immune response in germinal
centers. Cell, 67(6), pp.1121-1129.
Blazer, V., Wolke, R., Brown, J. and Powell, C., 1987. Piscine macrophage aggregate
parameters as health monitors: effect of age, sex, relative weight, season and site quality in
largemouth bass (Micropterus salmoides). Aquatic Toxicology, 10(4), pp.199-215.
Brattgjerd, S. and Evensen, Ø., 1996. A Sequential Light Microscopic and Ultrastructural
Study on the Uptake and Handling of Vibrio salmonicida in Phagocytes of the Head Kidney in
Experimentally Infected Atlantic Salmon (Salmo salar L.). Veterinary Pathology, 33(1), pp.5565.
Diaz-Satizabal, L. and Magor, B., 2015. Isolation and cytochemical characterisation of
melanomacrophages and melanomacrophage clusters from goldfish (Carassius auratus,
L.). Developmental & Comparative Immunology, 48(1), pp.221-228.
Fabacher, D. and Baumann, P., 1985. Enlarged livers and hepatic microsomal mixed-function
oxidase components in tumor-bearing brown bullheads from a chemically contaminated
river. Environmental Toxicology and Chemistry, 4(5), pp.703-710.
Fagarasan, S., 2000. T-Independent Immune Response: New Aspects of B Cell
Biology. Science, 290(5489), pp.89-92.
Falk, K., Press, C., Landsverk, T. and Dannevig, B., 1995. Spleen and kidney of Atlantic salmon
(Salmo salar L.) show histochemical changes early in the course of experimentally induced
infectious salmon anaemia (ISA). Veterinary Immunology and Immunopathology, 49(1-2),
pp.115-126.
Ferguson, H., 1976. The relationship between ellipsoids and melano-macrophage centres in
the spleen of turbot (Scophthalmus maximus). Journal of Comparative Pathology, 86(3),
pp.377-380.
Fisheries Research and Development, 2019. Australian Fisheries And Aquaculture Industry
2017/18: Economic And Social Contributions Summary. p.2.
Haaparanta, A., Valtonen, E., Hoffmann, R. and Holmes, J., 1996. Do macrophage centres in
freshwater fishes reflect the differences in water quality?. Aquatic Toxicology, 34(3),
pp.253-272.
Heesters, B., Myers, R. and Carroll, M., 2014. Follicular dendritic cells: dynamic antigen
libraries. Nature Reviews Immunology, 14(7), pp.495-504.
Herraez, M. and Zapata, A., 1986. Structure and function of the melano-macrophage centres
of the goldfishCarassius auratus. Veterinary Immunology and Immunopathology, 12(1-4),
pp.117-126.
Herraez, M. and Zapata, A., 1987. Trapping of intraperitoneal-injected Yersinia ruckeri in the
lymphoid organs of Carassius auratus: the role of melano-macrophage centres. Journal of
Fish Biology, 31(sa), pp.235-237.
Johnson, J., Schwiesow, T., Ekwall, A. and Christiansen, J., 1999. Reptilian
Melanomacrophages Function under Conditions of Hypothermia: Observations on
Phagocytic Behavior. Pigment Cell Research, 12(6), pp.376-382.
Kelly-Reay, K. and Weeks-Perkins, B., 1994. Determination of the macrophage
chemiluminescent response in Fundulus heteroclitus as a function of pollution stress. Fish &
Shellfish Immunology, 4(2), pp.95-105.
Lamers, C., 1986. Histophysiology of a primary immune response against Aeromonas
hydrophila in carp (Cyprinus carpio L.). Journal of Experimental Zoology, 238(1), pp.71-80.
Lamers, C. and De Haas, M., 1985. Antigen localisation in the lymphoid organs of carp
(Cyprinus carpio). Cell and Tissue Research, 242(3), pp.491-498.
McHeyzer-Williams, L. and McHeyzer-Williams, M., 2005. Antigen-Specific Memory B-cell
development. Annual Review of Immunology, 23(1), pp.487-513.
Meinelt, T., Krüger, R., Pietrock, M., Osten, R. and Steinberg, C., 1997. Mercury pollution
and macrophage centres in pike (Esox lucius) tissues. Environmental Science and Pollution
Research, 4(1), pp.32-36.
Peters, G. and Schwarzer, R., 1985. Changes in hemopoietic tissue of rainbow trout under
influence of stress. Diseases of Aquatic Organisms, 1, pp.1-10.
Press, C., Dannevig, B. and Landsverk, T., 1994. Immune and enzyme histochemical
phenotypes of lymphoid and nonlymphoid cells within the spleen and head kidney of
Atlantic salmon (Salmo salar L.). Fish & Shellfish Immunology, 4(2), pp.79-93.
Saunders, H., Oko, A., Scott, A., Fan, C. and Magor, B., 2010. The cellular context of AID
expressing cells in fish lymphoid tissues. Developmental & Comparative Immunology, 34(6),
pp.669-676.
Sayed, A. and Younes, H., 2017. Melanomacrophage centers in Clarias gariepinus as an
immunological biomarker for toxicity of silver nanoparticles. Journal of Microscopy and
Ultrastructure, 5(2), p.97.
Secombes, C. and Manning, M., 1980. Comparative studies on the immune system of fishes
and amphibians: antigen localisation in the carp Cyprinus carpio L. Journal of Fish Diseases,
3(5), pp.399-412.
Solem, S. and Stenvik, J., 2006. Antibody repertoire development in teleosts—a review with
emphasis on salmonids and Gadus morhua L. Developmental & Comparative Immunology,
30(1-2), pp.57-76.
Stebegg, M., Kumar, S., Silva-Cayetano, A., Fonseca, V., Linterman, M. and Graca, L., 2018.
Regulation of the Germinal Center Response. Frontiers in Immunology, 9.
Stegeman, J., 1978. Influence of Environmental Contamination on Cytochrome P-450 MixedFunction Oxygenases in Fish: Implications for Recovery in the Wild Harbor Marsh. Journal of
the Fisheries Research Board of Canada, 35(5), pp.668-674.
Steinel, N. and Bolnick, D., 2017. Melanomacrophage Centers As a Histological Indicator of
Immune Function in Fish and Other Poikilotherms. Frontiers in Immunology, 8.
Victora, G. and Nussenzweig, M., 2012. Germinal Centers. Annual Review of Immunology,
30(1), pp.429-457.
Vigliano, F., Bermúdez, R., Quiroga, M. and Nieto, J., 2006. Evidence for melanomacrophage centres of teleost as evolutionary precursors of germinal centres of higher
vertebrates: An immunohistochemical study. Fish & Shellfish Immunology, 21(4), pp.467471.
Widdicombe, M., Power, C., Van Gelderen, R., Nowak, B. and Bott, N., 2020. Relationship
between Southern Bluefin Tuna, Thunnus maccoyii, melanomacrophage centres and
Cardicola spp. (Trematoda: Aporocotylidae) infection. Fish & Shellfish Immunology, 106,
pp.859-865.
Wolke, R., 1992. Piscine macrophage aggregates: A review. Annual Review of Fish Diseases,
2, pp.91-108.
Wolke, R., Murchelano, R., Dickstein, C. and George, C., 1985. Preliminary evaluation of the
use of macrophage aggregates (MA) as fish health monitors. Bulletin of Environmental
Contamination and Toxicology, 35(1), pp.222-227.
Yu, M., Kiley, C., Sarot, D. and Perlmutter, A., 1971. Relation of Hemosiderin to
Erythropoiesis in the Blue Gourami, Trichogaster trichopterus. Journal of the Fisheries
Research Board of Canada, 28(1), pp.47-48.