Demonstrating the Intracellularity of

Borrelia burgdorferi

A compilation by

Harold Smith MD

This has also been published in

The Lyme Times 2000 Spring;28:86-88 (in cache)
Lyme Disease Resource Center
P.O.Box 707
Weaverville, CA 96093 - 0707
USA

Contents

1. Borrelia possesses the capability of penetrating cell wall or junctions.
2. Internalization once in the skin
3. Uptake of Bb into cell
4. Invasion of our own lymphocytes
5. Bb often is not found with silver staining.
6. Antigens within vacuoles
7. Bb binds deeply into the cell membrane.
8. Bb survived for at least 14 days of abx exposure.
9. Bb present from healed site of erythema migrans.
10. Vacuolated Langerhans cells release debris of Bb.
11. Bb were discharged into human leukocytes cytoplasm.
12. Cells take up the spirochetes intracellularly with multiple abnormal patterns.
13. In human tissue these forms are the same appearance of what is seen in vitro culture.
14. Bb found intracellular in the perinuclear region within human fibroblasts.
15. Persistence of spirochetes within macrophages
16. The challenge of intracellular pathogens
       Summary

1. Borrelia possesses the capability of penetrating cell wall or junctions.

Burgdorf shows borrelia leaves the tick gut endothelium when faced with incoming blood meal (and plasminogen) by invading gut wall and spreading via hemolymph to salivary apparatus. Hu, Perides, Noring, Klempner (Inf and Imm Sept 1995:3491) reveal borrelia binds human plasminogen to surface proteins and when this is converted to plasmin it provides the mechanism by which Bb can digest extracellular matrix and penetrate cell walls using the hosts own enzymes. So Borrelia has the evolutionary practice and the equipment to penetrate.
 

2. Internalization once in the skin

Filgueira et al in J of Imm 1996 157:2998-3005 disclose by electron microscopy of human skin biopsy that blood borne and skin origin dendritic cells internalize the Bb into these cells. Some are located "free " in cytosols and others are in phagolysosomes (i.e. some are not killed and some are).
 

3. Uptake of Bb into cell

Ying Ma in Infection and Immunity Feb 1991 p671-78 used human umbilical cord endothelial cells in culture to take up Borrelia into the cell bodies.  Withing 24 hrs up to 25% of Bb was internalized. Thus "The DEMONSTRATION (authors words) of Bb within endothelial cells suggests that intracellular localization may be a potential mechanism by which the organism escapes from the immune response of the host and contributes to persistence of the organism during later stages of Lyme disease." Of course this is how we suspect Borrelia invades into cells- first into the vessel lining and then into the tissue cells.
 

4. Invasion of our own lymphocytes

Dorward et all in Clin Inf Diseases 1997:25 Suppl 1 S2-8, "Invasion and cytopathic killing of Human Lymphocytes by Spirochetes Causing Lyme Disease".  "We found that Bb actively attaches to, invades, and kills human B- and T- lymphocytes- significant killing began within 1 hour of mixing. The results suggest that invasion and lysis of lymphocytes may constitue previously unrecognized factors in Lyme disease."
 

5. Bb often is not found with silver staining.

Although Bb often is not found with silver staining, when tissue is studied by DNA analysis, Borrelia is present.  This is evidence that the Borreliae are intracellular and thus not seen with silver staining but found when tissue extracts are analyzed for Bb DNA.  Priem in Ann Rhem Dis 1998 Feb 57 118-21, "Detection of Bb by PCR in synovial membrane, but not in synovial fluid from patients with persisting Lyme arthritis after antibiotic therapy".
 

6. Antigens within vacuoles

in Human Pathol 1996 Oct 27 1025-34 Nanagara & Duray and collaborators used electron microscopy with immune features to localize spirochetal antigens within vacuoles of fibroblasts and cytophagosomes of mononuclear cells and suggest sites at which spirochetes may elude host immune response and antibiotic treatment.
 

7. Bb binds deeply into the cell membrane

in Molecular Medicine 1999. 5: 46-54 Dennis Grab et all provide fascinating evidence that in HUMAN fibrocytes cultured with Bb that Bb binds deeply into the cell membrane in a coiling fashion and thus is carried to sites of injury while at the same time protected from attack by the immune system
 

8. Bb survived for at least 14 days of abx exposure

This also may be why when human foreskin fibroblasts were cultured with Bb and then ceftriaxone was added, the Bb survived for at least 14 days of exposure but not when fibroblasts were absent.  Georgilis in  J Infect Dis 1992 Aug;166(2):440-4  state -obviously "Fibroblasts Protect the Lyme Disease Spirochete Bb from Ceftriazone in Vitro".  Antibiotics aren't working intracellularly.
 

9. Bb present from healed site of erythema migrans

Next we have isolation of Bb from biopsy specimens taken from healthy looking skin of patients with Lymeborreliosis  (Kuiper J Clin Microbiol 1994 Mar 32 .715-20).  Six patients have Bb present from healed site of EM up to 6 months later by culture and PCR but not by silver staining. Where is the Bb? ... could be intracellular.

Likewise in PCR reaction for detection of Bb DNA in skin lesions of early and late Lyme borreliosis von Stedingk in Eur J Clin Microbiol Infec Dis 1995 Jan ; 14 ;1-5 found that over 60% of these skin biopsies had Bb DNA and after antibiotic treatment were negative but staining could not demonstrate Bb.  Where were they?
 

10. Vacuolated Langerhans cells release debris of Bb.

Hulinska in Zentralbl Bakteriol 1994 Jan 280:349-59. using electron microscopy of human ECM found vacuolated Langerhans cells releasing debris of Bb into lymphocyte infiltrates.
 

11. Bb were discharged into human leukocytes cytoplasm.

Hulinska in JSTD 1995;2:82-86 showed with electron microscoy that Bb spirochetes were discharged into human leukocytes cytoplasm but where not surrounded by phagosomal membranes.  "This could be one of the possible mechanisms of persistence of Bb in the host organism."
 

12. Cells take up the spirochetes intracellularly with multiple abnormal patterns.

Rittig et al in J Pathol 1994 Jul:173(3) 269-82 incubates Human peripheral blood monocytes, leudoctes, and synovial macrophages with Bb and then with light and electron microscopy finds the cells take up the spirochetes intracellularly with multiple abnormal patterns such as leaky lysosomes, invagination of large membrane areas and formation of giant cells and clusters with Bb inside.  "Moreover, these results may provide new insights into the pathnogenesis of other infectious diseases characterized similarly by microbial persistence".
 

13. In human tissue these forms are the same appearance of what is seen in vitro culture.

Aberer et al. in Amer J of Dermatopathology 1996.18:571-579 use precise 3-dimensional video microscopy with immunohistochemical staining to demonstrate all the variant forms of Borrelia and that in human tissue these forms are the same appearance of what is seen in vitro cultures. At the same time Bb are seen in macrophages and keratinocytes of the skin intracellularly.
 

14. Bb found intracellular in the perinuclear region within human fibroblasts.

Klempner et al in J Infeft Dis 1993:167: 1074-81 uses human fibroblasts and electron microscopy to show Bb are attached to the cell surface of fibroblasts after 24 to 48 hours of incubation but are eliminated from the cell surface by 5 days of ceftriaxone.  Yet Bb are still culture viable from the lysates of fibroblasts and found intracellular in the perinuclear region within human fibroblasts by laser scanning confocal microscopy.  "These observations suggest that Bb can adhere to, penetrate, and invade human fibroblasts in organisms that remain viable".
 

15. Persistence of spirochetes within macrophages

Of course mice aren't human, but they are part of the chain of evidence from tick to mouse to tick to human- so we are allowed to apply logic in "Demonstration".  Malawista et al. in J  Immunol 1993 Feb 150:909-15 clearly demonstrate the Bb spirochetes living within macrophages by scanning confocal microscopoy at multiple times after infection. "Persistence of spirochetes within macrophages provides a possible pathogenetic mechanism for chronic or recurrent Lyme disease in man".
 

16.  The challenge of intracellular pathogens

Although a little out of date- Kathleen Seiler and Weis in Current Opinion in Immunology 1996 8:503-509 summarize "Immunity to Lyme diseaes: protection pathology and persistence". The authors make reference to intracellular location
 but one of the best summaries of intracellular concern is The Challenge of Intracellular Pathogens by Mahmoud in New England Journal of Medicine March 1992 Vol 326 No 11 pages 761-2 which outlines the dangers of intracellular replication.
 

Summary

In summary, there is ample evidence demonstrating intracellular pathology involving humans and Borrelia burgdorferi.  Science is only as good as its hypotheses- and it is time to hypothesize that a cure for Borreliosis in humans must involve intracellular eradication.  The autoimmunity of chronic borreliosis ( molecular mimicry) is itself evidence of intracellular location- when the organism is coated with the hosts own cell structures ( thyroglobulin, myelin, nuclear debris, cardiolipin, etc) and then recognized as partly microbe and partly self structure, the basis is set for persistent immune attack- due to intracellular location. Thus all the elevated antimyelin antibodies, anti thyroglobulin, anti DNA, anti cardiolipin etc.

References


Infect Immun 1995 Sep;63(9):3491-6
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Binding of human plasminogen to Borrelia burgdorferi.

Hu LT, Perides G, Noring R, Klempner MS

Division of Geographic Medicine and Infectious Diseases, Tufts University School of Medicine, New England Medical Center, Boston, Massachusetts 02111, USA.

We studied the binding of plasminogen to Borrelia burgdorferi, a spirochete which causes Lyme disease and produces no endogenous proteases which digest extracellular matrix proteins.
 

Plasminogen bound to the surface of B. burgdorferi could be converted into plasmin by a human urokinase-type plasminogen activator. 125I-labeled plasminogen binding to both the 70-kDa protein and Osp A was inhibited by approximately 90% with a 1,000-fold excess of unlabeled plasminogen. The association of host plasmin(ogen) with borrelial surface proteins provides a mechanism by which B. burgdorferi can digest extracellular matrix and disseminate.

PMID: 7642282, UI: 95369905

J Immunol 1996 Oct 1;157(7):2998-3005
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Human dendritic cells phagocytose and process Borrelia burgdorferi.

Filgueira L, Nestle FO, Rittig M, Joller HI, Groscurth P

Department of Anatomy, University of Zurich, Switzerland.

There is strong evidence that the immune response to Borrelia burgdorferi (Bb) contributes to the pathogenesis of Lyme disease. Bb are transmitted by ticks to the skin, which is particularly rich in dendritic cells (DC). The initial reaction of these APCs may already set the course to immune pathogenesis.

To study the role of dendritic cells,

  1. biopsies from human skin were incubated with Bb and investigated in toto with electron microscopy. In addition,
  2. dendritic cells freshly isolated from dermis (DDC) and epidermis (Langerhans cells) were compared with blood-derived dendritic cells (BDC).
In situ, Bb were found in the dermal layer of the skin only, occasionally cleared by DDC. readily engulfed Bb preferentially using coiling phagocytosis.
 
  1. Internalized Bb were located
    • free in the cytosol and
    • inside of phagolysosomes of DDC and BDC.
  2. Intravesicular Bb antigens were colocalized with MHC class II molecules.
  3. In addition, live Bb induced IL-12 production in BDC. Bb-pulsed BDC
    • activated naive and
    • primed autologous Bb-specific T cells,
    as measured by detection of granulocyte-macrophage CSF gene transcription and proliferative response, respectively.
These data indicate that human dendritic cells Bb antigens. The way in which dendritic cells may influence the immune response in Lyme disease, however, remains to be evaluated.

PMID: 8816408, UI: 96413246

Infect Immun 1991 Feb;59(2):671-8
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Intracellular localization of Borrelia burgdorferi within human endothelial cells.

Ma Y, Sturrock A, Weis JJ

Department of Pathology, University of Utah School of Medicine, Salt Lake City 84132.

The later stages of infection by the Lyme disease pathogen, Borrelia burgdorferi, are characterized by the persistence of the organism in individuals possessing a strong anti-Borrelia immune response. This suggests that the organism is sequestered in a tissue protected from the immune system of the host or there is a reservoir of the organism residing within the cells of the host. In this report, the ability of B. burgdorferi to gain entrance into human umbilical vein endothelial cells was explored as a model for invasion. Incubation of B. burgdorferi with human umbilical vein endothelial cells at ratios ranging from 200:1 to 5,000:1 resulted in the intracellular localization of 10 to 25% of B. burgdorferi in 24 h. The intracellular location of the spirochetes was demonstrated by the incorporation of radiolabeled B. burgdorferi into a trypsin-resistant compartment and was confirmed by double-immunofluorescence staining which differentiated intracellular from extracellular organisms. Actin-containing microfilaments were required for the intracellular localization, indicating that the host cell participates in the internalization process. Activation of endothelial cells by agents known to increase the expression of several adhesion molecules had no effect on the interaction of B. burgdorferi with the endothelial monolayer. This indicates that the endothelial receptor for B. burgdorferi is constitutively expressed and that internalization is not dependent upon adhesion molecules whose expression is induced by inflammatory mediators. The demonstration of B. burgdorferi within endothelial cells suggest that intracellular localization may be a potential mechanism by which the organism escapes from the immune response of the host and may contribute to persistence of the organism during the later stages of Lyme disease.

PMID: 1987083, UI: 91100043


Clin Infect Dis 1997 Jul;25 Suppl 1:S2-8
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Invasion and cytopathic killing of human lymphocytes by spirochetes causing Lyme disease.

Dorward DW, Fischer ER, Brooks DM

National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana 59840, USA.

Lyme disease is a persistent low-density spirochetosis caused by Borrelia burgdorferi sensu lato. Although spirochetes causing Lyme disease are highly immunogenic in experimental models, the onset of specific antibody responses to infection is often delayed or undetectable in some patients. The properties and mechanisms mediating such immune avoidance remain obscure. To examine the nature and consequences of interactions between Lyme disease spirochetes and immune effector cells, we coincubated B. burgdorferi with primary and cultured human leukocytes.

We found that B. burgdorferi actively attaches to, invades, and kills human B and T lymphocytes.

These results suggest that invasion and lysis of lymphocytes may constitute previously unrecognized factors in Lyme disease and bacterial pathogenesis.

PMID: 9233657, UI: 97376886

Ann Rheum Dis 1998 Feb;57(2):118-21
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Detection of Borrelia burgdorferi by polymerase chain reaction in synovial membrane, but not in synovial fluid from patients with persisting Lyme arthritis after antibiotic therapy.

Priem S, Burmester GR, Kamradt T, Wolbart K, Rittig MG, Krause A

Charite University Hospital, Department of Medicine III, Rheumatology and Clinical Immunology, Berlin, Germany.

OBJECTIVES: To identify possible sites of bacterial persistence in patients with treatment resistant Lyme arthritis. It was determined whether Borrelia burgdorferi DNA may be detectable by polymerase chain reaction (PCR) in synovial membrane (SM) when PCR results from synovial fluid (SF) had become negative after antibiotic therapy. METHODS: Paired SF and SM specimens and urine samples from four patients with ongoing or recurring Lyme arthritis despite previous antibiotic therapy were investigated. A PCR for the detection of B burgdorferi DNA was carried out using primer sets specific for the ospA gene and a p66 gene of B burgdorferi. RESULTS: In all four cases, PCR with either primer set was negative in SF and urine, but was positive with at least one primer pair in the SM specimens. In all patients arthritis completely resolved after additional antibiotic treatment. CONCLUSIONS: These data suggest that in patients with treatment resistant Lyme arthritis negative PCR results in SF after antibiotic therapy do not rule out the intraarticular persistence of B burgdorferi DNA. Therefore, in these patients both SF and SM should be analysed for borrelial DNA by PCR as positive results in SM are strongly suggestive of ongoing infection.

PMID: 9613343, UI: 98276291

Hum Pathol 1996 Oct;27(10):1025-34
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Ultrastructural demonstration of spirochetal antigens in synovial fluid and synovial membrane in chronic Lyme disease: possible factors contributing to persistence of organisms.

Nanagara R, Duray PH, Schumacher HR Jr

Allergy-Immunology-Rheumatology Division, Department of Medicine, Faculty of Medicine, KhonKaen University, Thailand.

To perform the first systematic electronmicroscopic (EM) and immunoelectron microscopy (IEM) study of the pathological changes and the evidence of spirochete presence in synovial membranes and synovial fluid (SF) cells of patients with chronic Lyme arthritis. EM examination was performed on four synovial membrane and eight SF cell samples from eight patients with chronic Lyme disease. Spirochetal antigens in the samples were sought by IEM using monoclonal antibody to Borrelia burgdorferi outer surface protein A (OspA) as the immunoprobe. Prominent ultrastructural findings were surface fibrin-like material, thickened synovial lining cell layer and signs of vascular injury. Borrelia-like structures were identified in all four synovial membranes and in two of eight SF cell samples. The presence of spirochetal antigens was confirmed by IEM in all four samples studied (one synovial membrane and three SF cell samples). OspA labelling was in perivascular areas, deep synovial stroma among collagen bundles, and in vacuoles of fibroblasts in synovial membranes; and in cytophagosomes of mononuclear cells in SF cell samples. Electron microscopy adds further evidence for persistence of spirochetal antigens in the joint in chronic Lyme disease. Locations of spirochetes or spirochetal antigens both intracellulary and extracellulary in deep synovial connective tissue as reported here suggest sites at which spirochaetes may elude host immune response and antibiotic treatment.

PMID: 8892586, UI: 97047745


Mol Med 1999 Jan;5(1):46-54
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Interaction of Borrelia burgdorferi with peripheral blood fibrocytes, antigen-presenting cells with the potential for connective tissue targeting.

Grab DJ, Lanners H, Martin LN, Chesney J, Cai C, Adkisson HD, Bucala R

Tulane Regional Primate Research Center, Covington, Louisiana, USA. grab@cc.saga-u.ac.jp

BACKGROUND: Borrelia Burgdorferi has a predilection for collagenous tissue and can interact with fibronectin and cellular collagens. While the molecular mechanisms of how B. burgdorferi targets connective tissues and causes arthritis are not understood, the spirochetes can bind to a number of different cell types, including fibroblasts. A novel circulating fibroblast-like cell called the peripheral blood fibrocyte has recently been described. Fibrocytes express collagen types I and III as well as fibronectin. Besides playing a role in wound healing, fibrocytes have the potential to target to connective tissue and the functional capacity to recruit, activate, and present antigen to CD4(+) T cells. MATERIALS AND METHODS: Rhesus monkey fibrocytes were isolated and characterized by flow cytometry. B. burgdorferi were incubated with human or monkey fibrocyte cultures in vitro and the cellular interactions analyzed by light and electron microscopy. The two strains of B. burgdorferi studied included JD1, which is highly pathogenic for monkeys, and M297, which lacks the cell surface OspA and OspB proteins. RESULTS: In this study, we demonstrate that B. burgdorferi binds to both human and monkey (rhesus) fibrocytes in vitro. This process does not require OspA or OspB. In addition, the spirochetes are not phagocytosed but are taken into deep recesses of the cell membrane, a process that may protect them from the immune system. CONCLUSIONS: This interaction between B. burgdorferi and peripheral blood fibrocytes provides a potential explanation for the targeting of spirochetes to joint connective tissue and may contribute to the inflammatory process in Lyme arthritis.

PMID: 10072447, UI: 99172272

J Infect Dis 1992 Aug;166(2):440-4
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Fibroblasts protect the Lyme disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro.

Georgilis K, Peacocke M, Klempner MS

Department of Medicine, New England Medical Center, Boston, Massachusetts.

The Lyme disease spirochete, Borrelia burgdorferi, can be recovered long after initial infection, even from antibiotic-treated patients, indicating that it resists eradication by host defense mechanisms and antibiotics. Since B. burgdorferi first infects skin, the possible protective effect of skin fibroblasts from an antibiotic commonly used to treat Lyme disease, ceftriaxone, was examined. Human foreskin fibroblasts protected B. burgdorferi from the lethal action of a 2-day exposure to ceftriaxone at 1 microgram/mL, 10-20 x MBC. In the absence of fibroblasts, organisms did not survive. Spirochetes were not protected from ceftriaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone. Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective effect. Thus, several eukaryotic cell types provide the Lyme disease spirochete with a protective environment contributing to its long-term survival.

PMID: 1634816, UI: 92340959

J Clin Microbiol 1994 Mar;32(3):715-20
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Isolation of Borrelia burgdorferi from biopsy specimens taken from healthy-looking skin of patients with Lyme borreliosis.

Kuiper H, van Dam AP, Spanjaard L, de Jongh BM, Widjojokusumo A, Ramselaar TC, Cairo I, Vos K, Dankert J

Department of Medical Microbiology, Academic Medical Centre, University Hospital, University of Amsterdam, The Netherlands.

Erythematous skin lesions due to infection with Borrelia burgdorferi will often disappear without antibiotic treatment. The aim of the study was to assess whether after disappearance of the erythematous skin lesion B. burgdorferi is still present in the healthy-looking skin of untreated patients. In six patients, a skin biopsy specimen was taken at the site of a previous erythematous skin lesion 1 to 6 months after disappearance of the lesion. Four of them presented with early disseminated Lyme borreliosis. In one additional patient with early disseminated Lyme borreliosis, the site of a previous tick bite was biopsied. None of these patients had been treated with antibiotics before presentation. The cultures of the skin biopsy specimens of the seven patients showed growth of Borrelia species. By rRNA gene restriction analysis and genospecies-specific PCR, six isolates were classified as Borrelia garinii and one as Borrelia group VS461. These results show that B. burgdorferi can still be cultured from the skin after disappearance of the erythematous skin lesion or at the site of a previous tick bite.

PMID: 8195384, UI: 94253337

Eur J Clin Microbiol Infect Dis 1995 Jan;14(1):1-5
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Polymerase chain reaction for detection of Borrelia burgdorferi DNA in skin lesions of early and late Lyme borreliosis.

von Stedingk LV, Olsson I, Hanson HS, Asbrink E, Hovmark A

Department of Clinical Microbiology, Karolinska Hospital, Stockholm, Sweden.

The aim of this study was to evaluate the polymerase chain reaction (PCR) as a diagnostic tool for Lyme borreliosis on large numbers of samples from clinically well-defined cases of early and late cutaneous borreliosis. Skin biopsy specimens from patients with erythema migrans and acrodermatitis chronica atrophicans were analysed blindly together with an equal number of control biopsies. Using two different dilutions of each DNA specimen increased the number of total positives detected. All of the 76 control biopsies were PCR negative. Biopsy specimens from 18 of 26 (69%) erythema migrans lesions and from 22 of 36 (61%) acrodermatitis chronica atrophicans lesions were PCR positive. Fourteen post-therapy biopsies from patients with acrodermatitis chronica atrophicans were all negative, supporting the opinion that antibiotic therapy is successful in this chronic manifestation of Lyme borreliosis.

PMID: 7729446, UI: 95246765

Zentralbl Bakteriol 1994 Jan;280(3):348-59
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Electron microscopy of Langerhans cells and Borrelia burgdorferi in Lyme disease patients.

Hulinska D, Bartak P, Hercogova J, Hancil J, Basta J, Schramlova J

Institute of Public Health, Prague, Czech Republic.

To investigate dermal and epidermal involvement in the presence of Borrelia burgdorferi and to analyze the role of Langerhans cells and keratinocytes, 14 cases of erythema chronicum migrans and two controls were studied by means of electron microscopy, using negative staining and sectioning techniques. Using immunoelectron microscopy and histochemistry, positive results for B. burgdorferi were disclosed in 5 cases of erythema chronicum migrans and 3 cases of neuroborreliosis which were confirmed by cultivation. We cultured 4 stains of B. burgdorferi from the skin, 1 from blood and 2 from cerebrospinal fluid in BSK medium. Near to the centre of erythema chronicum migrans with focal necrosis were both a dissolved basal membrane and keratinocyte desmosomes surrounding damaged B. burgdorferi cells in the epidermis. Markedly oedematous keratinocytes and Langerhans cells with B. burgdorferi were released into lymphocyte infiltrates. At the periphery of all erythema chronicum migrans lesions, keratinocytes were well preserved while all dendritic cells seemed to be vacuolated. Above foci of B. burgdorferi located perivascular or among collagen fibers, Langerhans cells were frequent and more granulated. The possible role of Langerhans cells in the identification and elimination of B. burgdorferi is discussed.

PMID: 8167429, UI: 94220807


Journal of Spirochetal and Tick-Borne Diseases 2(4):82-86, 1995.
© 1995 Lyme Disease Foundation, Inc.

Intracellular Morphological Events Observed by Electron Microscopy on Neutrophil Phagocytosis of Borrelia garinii

Dagmar Hulinska, PhD, Jiri Basta, Rossella Murgia, Marina Cinco, MSc

Electron microscopy was used to present morphological events, which accompany the uptake of Borrelia garinii by polymorphonuclear leukocytes (PMN) and intracellular events of phagocytosis throughout 2 hours of contact. Phagocytosis of borreliae proceeded very quickly, and seemed to be independent of opsonins. Opsonized borreliae emitted membrane-bound blebs, which were attached and engulfed by PMN. Evidence of coiling and conventional phagocytosis were reported in the same cell. Coiling phagocytosis was time dependent (number of spirochetes internalized by this mechanism with time increased), but was not the preferential mechanism of engulfment by borreliae. Borreliae internalized by coiling phagocytosis were less morphologically altered, and their flagellae protruded into the host cytoplasm. The spirochetes were found discharged in PMN cytoplasm, and were not surrounded by a phagosomal membrane. This could be one of the possible mechanism of persistence of Borrelia burgdorferi in the host organism.

http://www.medscape.com/SLACK/JSTD/1995/v02.n04/jst0204.03.huli/pnt-jst0204.03.huli.html

J Pathol 1994 Jul;173(3):269-82
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Borrelia burgdorferi-induced ultrastructural alterations in human phagocytes: a clue to pathogenicity?

Rittig MG, Haupl T, Krause A, Kressel M, Groscurth P, Burmester GR
thomas.haeupl@rz.hu-berlin.de

Department of Anatomy I, University of Erlangen, Germany.

A chronic infection with the spirochaete Borrelia burgdorferi typically results in a multistage, multisystem illness. Thus, Lyme borreliosis may provide an interesting model to study the pathomechanisms of microbial persistence. In the present investigation, human peripheral blood monocytes, polymorphonuclear leukocytes, and synovial macrophages were incubated with B. burgdorferi and examined by light and electron microscopy. It was found that incubation with the spirochaetes induced distinct features in the phagocytes. Features which may be related to the pathogenesis of Lyme disease included the segmental uptake of spirochaetes with leaky lysosomes, the invagination of large membrane areas, the extra-lysosomal degradation of internalized B. burgdorferi cells and, finally, the formation of mononuclear syncytial cells and homotypic cell clusters. Features of unknown relevance were the occurrence of two types of cytoplasmic inclusion bodies and exocytic vesicles. These novel findings suggest that reactive alterations of the phagocytes may contribute to the pathogenesis of Lyme borreliosis, which could help to focus future histopathological studies. Moreover, these results may provide new insights into the pathogenesis of other infectious diseases characterized similarly by microbial persistence.

Am J Dermatopathol 1996 Dec;18(6):571-9
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Heterogeneity of Borrelia burgdorferi in the skin.

Aberer E, Kersten A, Klade H, Poitschek C, Jurecka W

Department of Dermatology, University of Vienna, Austria.

The reliability of various in vitro techniques to identify Borrelia burgdorferi infection is still unsatisfactory. Using a high-power resolution videomicroscope and staining with the borrelia genus-specific monoclonal flagellar antibody H9724, we identified borrelial structures in skin biopsies of erythema chronicum migrans (from which borrelia later was cultured), of acrodermatitis chronica atrophicans, and of morphea. In addition to typical borreliae, we noted stained structures of varying shapes identical to borreliae found in a "borrelia-injected skin" model; identical to agar-embedded borreliae; and identical to cultured borreliae following exposure to hyperimmune sera and/or antibiotics. We conclude that the H9724-reactive structures represent various forms of B. burgdorferi rather than staining artifacts. These "atypical" forms of B. burgdorferi may represent in vivo morphologic variants of this bacterium.

PMID: 8989928, UI: 97144094

J Infect Dis 1993 May;167(5):1074-81
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Invasion of human skin fibroblasts by the Lyme disease spirochete, Borrelia burgdorferi.

Klempner MS, Noring R, Rogers RA

The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell surface by treatment with ceftriaxone (1 microgram/mL) for 5 days. Despite the absence of visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear region within human fibroblasts by laser scanning confocal microscopy. Intracellular spirochetes specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent laser scanning confocal microscopy. These observations suggest that B. burgdorferi can adhere to, penetrate, and invade human fibroblasts in organisms that remain viable.

PMID: 8486939, UI: 93253286

J Immunol 1993 Feb 1;150(3):909-15
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The fate of Borrelia burgdorferi, the agent for Lyme disease, in mouse macrophages. Destruction, survival, recovery.

Montgomery RR, Nathanson MH, Malawista SE

Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510.

The macrophage is a known reservoir for a number of infectious agents, and is therefore a likely candidate site for persistence of Borrelia burgdorferi, the Lyme spirochete. We report that unopsonized B. burgdorferi enter macrophages rapidly, resulting mainly in degradation but occasionally in apparent intracellular persistence. We studied uptake of spirochetes by macrophages by simultaneously labeling infected cells with antibodies to B. burgdorferi and with sequential components of the endocytic pathway, and we examined optical sections (0.5-1.0 micron in thickness) of these cells by confocal fluorescence microscopy at multiple time points after infection. We found that only 5 min of incubation at 37 degrees C were required for nearly 100% of B. burgdorferi to enter a lysosomal glycoprotein-positive compartment, whereas 60 min were required for 90% of the spirochetes to appear in a cathepsin L-positive compartment under the same conditions. We also labeled infected living cells with acridine orange to distinguish live from killed intracellular organisms. Although the large majority of spirochetes within a given cell were dead, we saw occasional live ones up to 24 h (the longest interval examined) after all extracellular organisms had been lysed in distilled water. Moreover, we can reculture spirochetes from macrophages after infection. Persistence of spirochetes within macrophages provides a possible pathogenetic mechanism for chronic or recurrent Lyme disease in man.

PMID: 8423346, UI: 93139523


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